技术资料

Amyotrophic lateral sclerosis (ALS) involves motor neuron death due to mislocalized TDP-43. Pathologic TDP-43 associates with stress granules (SGs),and lowering the SG-associated protein ataxin-2 (ATXN2) using Atxn2-targeting antisense oligonucleotides prolongs survival in TAR4/4 sporadic ALS mice but failed in clinical trials likely due to poor target engagement. Here we show that an AAV with potent motor neuron transduction delivering Atxn2-targeting miRNAs reduces Atxn2 throughout the central nervous system at doses 40x lower than published work. In TAR4/4 mice,miAtxn2 increased survival (50%) and strength,and reduced motor neuron death,inflammation,and phosphorylated TDP-43. TAR4/4 transcriptomic dysregulation recapitulated ALS gene signatures that were rescued by miAtxn2,identifying potential therapeutic mechanisms and biomarkers. In slow progressing hemizygous mice,miAtxn2 slowed disease progression,and in ALS patient-derived lower motor neurons,our AAV vector transduced >95% of cells and potently reduced ATXN2 at MOI 4 logs lower than previously reported. These data support AAV-RNAi targeting ATXN2 as a translatable therapy for sporadic ALS. Amado et al. develop a gene therapy for sporadic ALS using motor neuron-targeting AAVs to deliver RNAi targeting ataxin-2. In a mouse model,survival,strength,and disease-related pathology are improved; and human motor neurons are strongly transduced. 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

Adherent two-dimensional human gastruloids have provided insights into early human embryogenesis. Even though the model system is highly reproducible,no available automated technology can screen and sort large numbers of these near-millimeter-sized complex structures for large-scale assays. Here,we developed a microraft array-based technology to perform image-based assays of large numbers of fixed or living gastruloids and sort individual gastruloids for downstream assays,such as gene expression analysis. Arrays of 529 indexed magnetic microrafts each (789?µm side length) possessing flat surfaces were photopatterned with a central circular region (500?µm diameter) of extracellular matrix with an accuracy of 93?±?1% to form a single gastruloid on each raft. An image analysis pipeline extracted features from transmitted light and fluorescence images of the gastruloids. The large microrafts were released and collected by an automated sorting system with efficiencies of 98?±?4% and 99?±?2%,respectively. The microraft array platform was used to assay individual euploid and aneuploid (possessing abnormal numbers of chromosomes) gastruloids with clear phenotypic differences. Aneuploid gastruloids displayed significantly less DNA/area than euploid gastruloids. However,even gastruloids with the same condition displayed significant heterogeneity. Both noggin (NOG) and keratin 7 (KRT7),two genes involved in spatial patterning within gastruloids,were upregulated in aneuploid relative to that in the euploid gastruloids. Moreover,relative NOG and KRT7 expressions were negatively correlated with DNA/area. The microraft arrays will empower novel screens of single gastruloids for a better understanding of key mechanisms underlying phenotypic differences between gastruloids. View Publication

BackgroundChronic lung disease of prematurity,called bronchopulmonary dysplasia (BPD),lacks effective therapies,stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD,but current protocols do not accurately replicate the distal niche environment of the native lung. Herein,we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation.MethodsHuman PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry,immunofluorescence,and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6),E-cadherin (CDH1),NK2 Homeobox 1 (NKX2-1),HT2-280,surfactant proteins B (SFTPB) and C (SFTPC).ResultsWe observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally,we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids,mimicking in situ fetal respiratory movements,increased AEC2 differentiation without affecting proximal epithelial differentiation.ConclusionOur data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03890-2. View Publication

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However,the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis,potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing,which will ultimately increase editing efficiency. In our study,we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines. View Publication

Coronary microvascular disease (CMD) and its progression towards major adverse coronary events pose a significant health challenge. Accurate in vitro investigation of CMD requires a robust cell model that faithfully represents the cells within the cardiac microvasculature. Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) offer great potential; however,they are traditionally derived via differentiation protocols that are not readily scalable and are not specified towards the microvasculature. Here,we report the development and comprehensive characterisation of a scalable 3D protocol enabling the generation of phenotypically stable cardiac hPSC-microvascular-like ECs (hPSC-CMVECs) and cardiac pericyte-like cells. These were derived by growing vascular organoids within 3D stirred tank bioreactors and subjecting the emerging 3D hPSC-ECs to high-concentration VEGF-A treatment (3DV). Not only did this promote phenotypic stability of the 3DV hPSC-ECs; single cell-RNA sequencing (scRNA-seq) revealed the pronounced expression of cardiac endothelial- and microvascular-associated genes. Further,the generated mural cells attained from the vascular organoid exhibited markers characteristic of cardiac pericytes. Thus,we present a suitable cell model for investigating the cardiac microvasculature as well as the endothelial-dependent and -independent mechanisms of CMD. Moreover,owing to their phenotypic stability,cardiac specificity,and high angiogenic potential,the cells described within would also be well suited for cardiac tissue engineering applications.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09929-5. View Publication

Microglia play a pivotal role in neurodegenerative disease pathogenesis,but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia,we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs),termed iMGs,harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism,autophagy dysregulation and deficient phagocytosis,a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P,a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways,as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders. Mutations in profilin 1 (PFN1),which modulates actin dynamics,are associated with ALS. Here the authors show that expression of ALS-PFN1 is sufficient to induce deficits in human microglia-like cells,including impaired phagocytosis and lipid metabolism,and that gain-of-function interactions between ALS-PFN1 and PI3P may underlie these deficits. View Publication

Klotho,a well-known aging suppressor protein,has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids,its potential to mitigate neurodegenerative processes triggered by ?-amyloid remains underexplored. In this study,we utilised human induced pluripotent stem cells (iPSCs) engineered with a doxycycline-inducible system to overexpress KLOTHO and generated 2D cortical neuron cultures from these cells. These neurons were next exposed to pre-aggregated ?-amyloid 1–42 oligomers to model the neurotoxicity associated with Alzheimer’s disease. Our data reveal that upregulation of KLOTHO significantly reduced ?-amyloid-induced neuronal degeneration and apoptosis,as evidenced by decreased cleaved caspase-3 expression and preservation of axonal integrity. Additionally,KLOTHO overexpression prevented the loss of dendritic branching and mitigated reductions in axonal diameter,hallmark features of neurodegenerative pathology. These results highlight Klotho’s protective role against ?-amyloid-induced neurotoxicity in human cortical neurons and suggest that its age-related decline may contribute to neurodegenerative diseases such as Alzheimer’s disease. Our findings underscore the therapeutic potential of Klotho-based interventions in mitigating age-associated neurodegenerative processes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13041-025-01199-6. 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

Huntington disease (HD) is a neurodegenerative disease caused by the abnormal expansion of a polyglutamine tract resulting from a mutation in the HTT gene. Oxidative stress has been identified as a significant contributing factor to the development of HD and other neurodegenerative diseases,and targeting anti-oxidative stress has emerged as a potential therapeutic approach. CHCHD2 is a mitochondria-related protein involved in regulating cell migration,anti-oxidative stress,and anti-apoptosis. Although CHCHD2 is highly expressed in HD cells,its specific role in the pathogenesis of HD remains uncertain. We postulate that the up-regulation of CHCHD2 in HD models represents a compensatory protective response against mitochondrial dysfunction and oxidative stress associated with HD. To investigate this hypothesis,we employed HD mouse striatal cells and human induced pluripotent stem cells (hiPSCs) as models to examine the effects of CHCHD2 overexpression (CHCHD2-OE) or knockdown (CHCHD2-KD) on the HD phenotype. Our findings demonstrate that CHCHD2 is crucial for maintaining cell survival in both HD mouse striatal cells and hiPSCs-derived neurons. Our study demonstrates that CHCHD2 up-regulation in HD serves as a compensatory protective response against oxidative stress,suggesting a potential anti-oxidative strategy for the treatment of HD. View Publication

Sustained smouldering,or low-grade activation,of myeloid cells is a common hallmark of several chronic neurological diseases,including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However,how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here,using a multiomics approach,we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically,blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3. Blocking mitochondrial complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in vivo in an animal disease model. View Publication

Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However,the substantial variability between existing protocols for generating IM cells compromises their efficiency,reproducibility,and overall success,potentially hindering the utility of urogenital system organoids. Here,we examined the role of high levels of Nodal signaling and BMP activity,as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 ?M CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 ?M CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence,this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro,with potential applications in regenerative medicine,drug discovery,and disease modeling. View Publication