技术资料

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

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

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

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