技术资料

ABSTRACTThe paradoxical exacerbation of cellular injury and death during reperfusion remains a problem in the treatment of myocardial infarction. Mitochondrial dysfunction plays a key role in the pathogenesis of myocardial ischemia and reperfusion injury. Dysfunctional mitochondria can be removed by mitophagy,culminating in their degradation within acidic lysosomes. Mitophagy is pivotal in maintaining cardiac homeostasis and emerges as a potential therapeutic target. Here,we employed beating human engineered heart tissue (EHT) to assess mitochondrial dysfunction and mitophagy during ischemia and reperfusion simulation. Our data indicate adverse ultrastructural changes in mitochondrial morphology and impairment of mitochondrial respiration. Furthermore,our pH-sensitive mitophagy reporter EHTs,generated by a CRISPR/Cas9 endogenous knock-in strategy,revealed induced mitophagy flux in EHTs after ischemia and reperfusion simulation. The induced flux required the activity of the protein kinase ULK1,a member of the core autophagy machinery. Our results demonstrate the applicability of the reporter EHTs for mitophagy assessment in a clinically relevant setting. Deciphering mitophagy in the human heart will facilitate development of novel therapeutic strategies. Summary: Mitochondrial dysfunction and lysosomal degradation of mitochondria (mitophagy) is induced after ischemia and reperfusion simulation in human engineered heart tissue,as shown with an endogenous pH-sensitive mitophagy reporter. View Publication

Oxidative phosphorylation (OXPHOS) in the mitochondrial inner membrane is a therapeutic target in many diseases. Neural stem cells (NSCs) show progress in improving mitochondrial dysfunction in the central nervous system (CNS). However,translating neural stem cell-based therapies to the clinic is challenged by uncontrollable biological variability or heterogeneity,hindering uniform clinical safety and efficacy evaluations. We propose a systematic top-down design based on membrane self-assembly to develop neural stem cell-derived oxidative phosphorylating artificial organelles (SAOs) for targeting the central nervous system as an alternative to NSCs. We construct human conditionally immortal clone neural stem cells (iNSCs) as parent cells and use a streamlined closed operation system to prepare neural stem cell-derived highly homogenous oxidative phosphorylating artificial organelles. These artificial organelles act as biomimetic organelles to mimic respiration chain function and perform oxidative phosphorylation,thus improving ATP synthesis deficiency and rectifying excessive mitochondrial reactive oxygen species production. Conclusively,we provide a framework for a generalizable manufacturing procedure that opens promising prospects for disease treatment. Regulating oxidative phosphorylation and restoring redox homeostasis are crucial in neurological disorders. Here,the authors develop a top-down membrane self-assembly strategy to develop stem cell-derived artificial organelles (SAOs) that mimic mitochondrial oxidative phosphorylation without the risks associated with stem cell therapy. View Publication

The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length of sequencing to several kilobases,thereby facilitating the identification of alternative splicing events and isoform expressions. Recently,numerous computational tools for isoform detection using long-read sequencing data have been developed. Nevertheless,there remains a deficiency in comparative studies that systemically evaluate the performance of these tools,which are implemented with different algorithms,under various simulations that encompass potential influencing factors. In this study,we conducted a benchmark analysis of thirteen methods implemented in nine tools capable of identifying isoform structures from long-read RNA-seq data. We evaluated their performances using simulated data,which represented diverse sequencing platforms generated by an in-house simulator,RNA sequins (sequencing spike-ins) data,as well as experimental data. Our findings demonstrate IsoQuant as a highly effective tool for isoform detection with LRS,with Bambu and StringTie2 also exhibiting strong performance. These results offer valuable guidance for future research on alternative splicing analysis and the ongoing improvement of tools for isoform detection using LRS data. Recently,various computational tools have emerged for detecting mRNA isoforms using long-read sequencing data. Here,the authors systemically evaluate and compare the performance of these tools. View Publication

Simple SummaryHuman induced pluripotent stem cells hold great promise for treating neurological diseases. One of the biggest challenges,however,is the immune system: if transplanted cells are not a perfect match,the body may reject them. To overcome this,we aimed to create “off-the-shelf”,universal cells that could be safely used in anyone,without needing a matched donor. Using CRISPR-mediated gene editing tool,we deleted two key genes,B2M and CIITA,that are responsible for making proteins recognized by the immune system. Additionally,we engineered the cells to produce MIF,which helps protect against natural killer cell attacks. Overall,our study shows that combining MIF overexpression with the removal of B2M and CIITA can produce universal cells that avoid rejection by the immune system. This approach could help make stem cell therapies more widely available and effective for spinal cord injuries and other diseases. AbstractHuman induced pluripotent stem cells (iPSCs) offer immense potential as a source for cell therapy in spinal cord injury (SCI) and other diseases. The development of hypoimmunogenic,universal cells that could be transplanted to any recipient without requiring a matching donor could significantly enhance their therapeutic potential and accelerate clinical translation. To create off-the-shelf hypoimmunogenic cells,we used CRISPR-Cas9 to delete B2M (HLA class I) and CIITA (master regulator of HLA class II). Double-knockout (DKO) iPSC-derived neural progenitor cells (NPCs) evaded T-cell-mediated immune rejection in vitro and after grafting into the injured spinal cord of athymic rats and humanized mice. However,loss of HLA class I heightened susceptibility to host natural killer (NK) cell attack,limiting graft survival. To counter this negative effect,we engineered DKO NPCs to overexpress macrophage migration inhibitory factor (MIF),an NK cell checkpoint ligand. MIF expression markedly reduced NK cell-mediated cytotoxicity and improved long-term engraftment and integration of NPCs in the animal models for spinal cord injury. These findings demonstrate that MIF overexpression,combined with concurrent B2M and CIITA deletion,generates hiPSC neural derivatives that escape both T- and NK-cell surveillance. This strategy provides a scalable route to universal donor cells for regenerative therapies in SCI and potentially other disorders. View Publication

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents,and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue,in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas,but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here,we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells,and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method,new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity,suitable stability and biodistribution,with negligible toxicity to islets,qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-84574-y. View Publication

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however,their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study,we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles,which,with a capacity of up to 36 kb,can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version,both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency,with sustained transgene expression observed for up to 110 days post-transduction. Moreover,we found efficient transduction of photoreceptors and Müller glial cells,without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However,an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction,suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery,further investigations are required to assess their long-term safety and efficacy. View Publication

Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During the cell cycle,nuclear speckles dissolve following phosphorylation of their protein components. Here,we identify the PP1 family as the phosphatases that counteract kinase-mediated dissolution. PP1 overexpression increases speckle cohesion and leads to retention of mRNA within speckles and the nucleus. Using APEX2 proximity labeling combined with RNA-sequencing,we characterize the recruitment of specific RNAs. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm,particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increases with nuclear speckle cohesion,the ratios of most mRNA species to each other are constant,indicating non-selective retention. We further find that cellular responses to heat shock,oxidative stress,and hypoxia include changes to the phosphorylation and cohesion of nuclear speckles and to mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization. Here the authors study how interactions with nuclear speckles help localize mRNA in cells. They find that modifications of the proteins in these structures affects their cohesion and can modulate mRNA retention under stress. View Publication

Neutrophils are essential innate immune cells with unusual anti-microbial properties while dysfunctions of neutrophils lead to severe health problems such as lethal infections. Generation of neutrophils from human induced pluripotent stem cells (hiPSCs) is highly promising to produce off-the-shelf neutrophils for transfusion therapies. However,the anti-microbial potencies of hiPSCs derived neutrophils (iNEUs) remain less documented. Here,we develop a scalable approach to generate iNEUs in a chemical defined condition. iNEUs display typical neutrophil characters in terms of phagocytosis,migration,formation of neutrophil extracellular traps (NETs),etc. Importantly,iNEUs display a strong killing potency against various bacteria such as K.pneumoniae,P.aeruginosa,E.coli and S.aureus. Moreover,transfusions of iNEUs in mice with neutrophil dysfunction largely enhance their survival in lethal infection of different bacteria. Together,our data show that hiPSCs derived neutrophils hold strong anti-microbial potencies to protect severe infections under neutrophil dysfunction conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00227-z. View Publication

Autoimmune uveitis is a leading cause of severe vision loss,and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq,RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU),revealing that EAU causes broad retinal neuron degeneration and marker downregulation,and that Müller glia may act as antigen-presenting cells. Moreover,EAU immune response is primarily driven by Th1 cells,and results in dramatic upregulation of CC chemokines,especially CCL5,in the EAU retina. Accordingly,overexpression of CCR5,a CCL5 receptor,in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU,by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together,our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03134-3. View Publication

Neurological damage caused by peripheral nerve injury can be devastating and is a common neurological disorder that,along with muscle disorders,reduces the quality of life. Neural crest cells (NCCs) are a transient cell population that occurs during embryogenesis,can differentiate into various cells upon transplantation,and has potential therapeutic effects on neurological diseases. However,there are limitations to cell therapy,such as uncontrolled differentiation and tumor formation. Extracellular vesicles (EVs),which are non-cellular potential therapeutic candidates,are nanosized membrane-bound vesicles. Studies have been reported using starch cells derived from neural cells,such as neural stem cells,because they are involved in cell-to-cell communication and carry a variety of bioactive molecules. We investigated the effects of EVs isolated from NCCs on neuronal cell death and inflammation. Additionally,we overexpressed the nerve growth factor (NGF),which is involved in neural cell growth and proliferation,in NCCs to further investigate the effects of EVs containing NGF. NCCoe-NGF-EVs showed neuroprotective and regenerative properties by modulating inflammatory pathway,promoting Schwann cell activation,and enhancing remyelination. In vitro studies on NCCoe-NGF-EVs suppressed pro-inflammatory cytokines and reduced oxidative stress-induced neuronal apoptosis through NF-?B pathway inhibition and ERK,AKT signal activation. We also evaluated the effect of EVs on neuropathy by performing in vivo study. Our results suggest that NCCoe-NGF-EV had neuroprotective effects by reducing neuronal apoptosis and promoting neuronal proliferation based on neurite outgrowth and anti-inflammation effects treated with NCCoe-NGF-EVs. In addition,NCCoe-NGF-EVs were protected muscle loss caused by peripheral nerve injury. NCCoe-NGF-EV induced regeneration of damaged nerves and inhibited cell death through anti-inflammatory effects. This study suggests the potential of NGF-enriched EVs as non-cellular therapeutic platform for peripheral neuropathies and other neuroinflammatory disorders. View Publication

SummaryCardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage,which must be repaired by DNA damage response proteins. However,the impact of DNA damage on global cardiomyocyte protein abundance,and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control,and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing,chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD,such as arrhythmia are enriched in specific DOX-correlated modules,DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead,they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD,and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation. View Publication

PurposeDilated cardiomyopathy (DCM) is a prevalent form of heart failure with limited therapeutic options. This study explores a novel treatment strategy involving the delivery of exosome-derived miRNA-185-5p inhibitors encapsulated in liposomes,aiming to target cardiac tissue and alleviate myocardial apoptosis and cuproptosis in DCM.MethodsThe miRNA-185-5p inhibitor,identified in our previous study and extracted from exosomes,was encapsulated in liposomes functionalized with a cardiac-targeting peptide. This system was used in both in vitro and in vivo models of DCM induced by doxorubicin (DOX). We evaluated the effects of this treatment on cardiac function,apoptosis,cuproptosis,oxidative stress,and fibrosis using echocardiography,histological analysis,Western blotting,and biochemical assays.ResultsIn vitro experiments demonstrated that the Lipo@miR-185-5p inhibitor markedly attenuated apoptosis and cuproptosis in H9C2 cells and iPSC-derived cardiomyocytes,with a 42.6% reduction in apoptotic cell rates and over 50% downregulation of cuproptosis-related markers (both P < 0.01). In vivo,the targeted liposomal formulation significantly improved cardiac function in DOX-induced DCM mice,as evidenced by a 27.3% increase in left ventricular ejection fraction (LVEF) and a 36.5% reduction in myocardial fibrosis area (P < 0.01),along with enhanced survival. These findings underscore the therapeutic potential of this targeted delivery strategy for the treatment of dilated cardiomyopathy.ConclusionLipo@miR-185-5p inhibitor,utilizing exosome-derived miRNA and targeted liposomal delivery,effectively alleviates DCM-induced myocardial dysfunction. This approach represents a promising therapeutic strategy for cardiovascular diseases by targeting specific molecular mechanisms involved in heart failure. View Publication