技术资料

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

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

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

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

BackgroundThe epoxyeicosatrienoic acids (EETs) are derivatives of the arachidonic acid metabolism with anti-inflammatory activities. However,their efficacy is limited due to the rapid hydrolysis by soluble epoxide hydrolase (sEH). Inhibition of sEH has been shown to stabilize the EETs and reduce neuroinflammation in A? mouse models of Alzheimer’s disease (AD). However,the role of the sEH-EET signaling pathway in other CNS cell types and neurodegenerative conditions are less understood.MethodsHere we investigated the mechanisms and functional role of the sEH-EET axis in tauopathy by treating PS19 mice with a small molecule sEH inhibitor TPPU and by crossing the PS19 mice with Ephx2 (gene encoding sEH) knockout mice. This was followed by single-nucleus RNA-sequencing (snRNA-seq),biochemical and immunohistochemical analysis,and behavioral assessments. Additionally,we examined the effects of the sEH-EET pathway in primary microglia cultures and human induced pluripotent stem cell (iPSC)-derived neurons exhibiting seeding-induced Tau inclusions.ResultssEH inhibition improved cognitive function,rescued neuronal cell loss,and reduced Tau pathology and microglial reactivity. snRNA-seq revealed that TPPU treatment upregulated genes involved in actin cytoskeleton and excitatory synaptic pathways. Treatment of human iPSC-derived neurons with TPPU enhanced synaptic density without affecting Tau accumulation,suggesting a cell-autonomous neuroprotective effect of sEH blockade. Furthermore,sEH inhibition reversed disease-associated and interferon-responsive microglial states in PS19 mice,while EET supplementation promoted Tau phagocytosis and clearance in primary microglia cultures.ConclusionThese findings demonstrate that sEH blockade or EET augmentation confers therapeutic benefit in neurodegenerative tauopathies by simultaneously targeting neuronal and microglial pathways.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00844-x. View Publication

Blood transfusion plays a vital role in modern medicine,but frequent shortages occur. Ex vivo manufacturing of red blood cells (RBCs) from universal donor cells offers a potential solution,yet the high cost of recombinant cytokines remains a barrier. Erythropoietin (EPO) signaling is crucial for RBC development,and EPO is among the most expensive media components. To address this challenge,we develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) using design-build-test cycles and genome editing. By integrating synEPOR at the endogenous EPOR locus in O-negative induced pluripotent stem cells,we achieve equivalent erythroid differentiation,transcriptomic changes,and hemoglobin production using small molecules compared to EPO-supplemented cultures. This approach dramatically reduces culture media costs. Our strategy not only addresses RBC production challenges but also demonstrates how protein and genome engineering can introduce precisely regulated cellular behaviors,potentially improving scalable manufacturing of a wide range of clinically relevant cell types. Shortages of donor blood for transfusions can have severe medical consequences,and ex vivo production of red blood cells offers a potential solution. Here authors developed synthetic EPO receptors,which allow erythropoiesis (red blood cell production) without the need for expensive EPO. View Publication

This study describes an OTULIN-related autoinflammatory syndrome (ORAS) patient with two rare heterozygous variants of OTULIN (p.P152L and p.R306Q); the latter is a de novo variant that acts in a dominant-negative manner to cause ORAS. OTULIN-related autoinflammatory syndrome (ORAS),a severe autoinflammatory disease,is caused by biallelic pathogenic variants of OTULIN,a linear ubiquitin-specific deubiquitinating enzyme. Loss of OTULIN attenuates linear ubiquitination by inhibiting the linear ubiquitin chain assembly complex (LUBAC). Here,we report a patient who harbors two rare heterozygous variants of OTULIN (p.P152L and p.R306Q). We demonstrated accumulation of linear ubiquitin chains upon TNF stimulation and augmented TNF-induced cell death in mesenchymal stem cells differentiated from patient-derived iPS cells,which confirms that the patient has ORAS. However,although the de novo p.R306Q variant exhibits attenuated deubiquitination activity without reducing the amount of OTULIN,the deubiquitination activity of the p.P152L variant inherited from the mother was equivalent to that of the wild-type. Patient-derived MSCs in which the p.P152L variant was replaced with wild-type also exhibited augmented TNF-induced cell death and accumulation of linear chains. The finding that ORAS can be caused by a dominant-negative p.R306Q variant of OTULIN furthers our understanding of disease pathogenesis. Graphical Abstract View Publication

Enhancing RuvBL1,but particularly RuvBL2 expression,reduces toxic dipeptide repeat proteins in vitro and in vivo models of C9orf72-linked ALS/FTD,suggesting that modulating RuvBL1/2 levels could be a promising therapeutic approach for C9ALS/FTD. A G4C2 hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Bidirectional transcription and subsequent repeat-associated non-AUG (RAN) translation of sense and antisense transcripts leads to the formation of five dipeptide repeat (DPR) proteins. These DPRs are toxic in a wide range of cell and animal models. Therefore,decreasing RAN-DPRs may be of therapeutic benefit in the context of C9ALS/FTD. In this study,we found that C9ALS/FTD patients have reduced expression of the AAA+ family members RuvBL1 and RuvBL2,which have both been implicated in aggregate clearance. We report that overexpression of RuvBL1,but to a greater extent RuvBL2,reduced C9orf72-associated DPRs in a range of in vitro systems including cell lines,primary neurons from the C9-500 transgenic mouse model,and patient-derived iPSC motor neurons. In vivo,we further demonstrated that RuvBL2 overexpression and consequent DPR reduction in our Drosophila model was sufficient to rescue a number of DPR-related motor phenotypes. Thus,modulating RuvBL levels to reduce DPRs may be of therapeutic potential in C9ALS/FTD. View Publication

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However,we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis,particularly in the late stage,where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus,we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge,the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this,we generated inducible TRIB3 overexpression hESCs,named TRIB3tet-on OE H9,based on a Tet-On system. Then,we analyzed hemoglobin expression,condensed chromosomes,organelle clearance,and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry,enhanced hemoglobin and related protein expression in Western blot,decreased nuclear area size,promoted enucleation rate,decreased lysosome and mitochondria number,more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 ?g/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore,our study delineates the role of TRIB3 in terminal erythropoiesis,and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin. Highlights•TRIB3 boosts erythroid cell maturation.•Key insights into erythropoiesis from hESCs.•Enhanced ubiquitin-proteasome system and downstream mitophagy in erythroid differentiation. View Publication

Efficient tumor T-cell infiltration is crucial for the effectiveness of T-cell-based therapies against solid tumors. Eosinophils play crucial roles in recruiting T cells in solid tumors. Our group has previously generated induced eosinophils (iEOs) from human pluripotent stem cells and exhibited synergistic efficacy with CAR-T cells in solid tumor inhibition. However,administrated eosinophils might influx into inflammatory lungs,posing a potential safety risk. Mitigating the safety concern and enhancing efficacy is a promising development direction for further application of eosinophils.MethodsWe developed a new approach to generate eosinophils with enhanced potency from human chemically reprogrammed induced pluripotent stem cells (hCiPSCs) with the Toll-like receptor (TLR) 7/8 signaling agonist R848.ResultsR848-activated iEOs (R-iEOs) showed significantly decreased influx to the inflamed lungs,indicating a lower risk of causing airway disorders. Furthermore,these R-iEOs had enhanced anti-tumor functions,preferably accumulated at tumor sites,and further increased T-cell infiltration. The combination of R-iEOs and CAR-T cells suppressed tumor growth in mice. Moreover,the chemo-trafficking signaling increased in R-iEOs,which may contribute to the decreased lung influx of R-iEOs and the increased tumor recruitment of T cells.ConclusionOur study provides a novel approach to alleviate the potential safety concerns associated with eosinophils while increasing T-cell infiltration in solid tumors. This finding offers a prospective strategy for incorporating eosinophils to improve CAR-T-cell immunotherapy for solid tumors in the future. View Publication

Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol,we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination-mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array,efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase,permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences,no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects. Key features • A single-vector system to deliver Cpf1 and crRNA enables the sorting of transfected cells • Efficient and simultaneous multi-modular genome editing exemplified by mutation of MAFB and knockin of AAVS1 loci in a single experiment • Edited PSCs showed minimal off-target effects and can be differentiated into multiple cell types. View Publication

Human embryonic stem cells (hESCs) serve as a valuable in vitro model for studying early human developmental processes due to their ability to differentiate into all three germ layers. Here,we present a comprehensive multi-omics dataset generated by differentiating hESCs into cardiomyocytes via the mesodermal lineage,collecting samples at 10 distinct time points. We measured mRNA levels by mRNA sequencing (mRNA-seq),translation levels by ribosome profiling (Ribo-seq),and protein levels by quantitative mass spectrometry-based proteomics. Technical validation confirmed high quality and reproducibility across all datasets,with strong correlations between replicates. This extensive dataset provides critical insights into the complex regulatory mechanisms of cardiomyocyte differentiation and serves as a valuable resource for the research community,aiding in the exploration of mammalian development and gene regulation. View Publication