技术资料

Inflammatory cytokines,including interleukin 6 (IL6),are associated with ion channel remodeling and enhance the propensity to alterations in cardiac rhythm generation and propagation,in which the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role. Hence,we investigated the consequences of exposure to IL6 on HCN channels in cell models and human atrial biopsies. In murine atrial HL1 cells and in cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs),IL6 elicited STAT3 phosphorylation,a receptor-mediated downstream signaling. Downregulation of HCN1,2,4 by IL6 was observed after 24–48 h; in hiPS-CMs,this effect was reverted by 24 h of application of tocilizumab,a human IL6 receptor antagonist. In parallel,hiPS-CM action potentials (APs) showed a reduced spontaneous frequency. Moreover,we assessed IL6 and HCN expression in dilated left atrial samples from patients with mitral valve disease,an AF-prone condition. IL6 levels were increased in dilated atria compared to controls and positively correlated with echocardiographic atrial dimensions. Interestingly,the highest IL6 transcript levels and the lowest HCN4 and HCN2 expression were in these samples. In conclusion,our data uncovered a novel link between IL6 and cardiac HCN channels,potentially contributing to atrial electrical disturbances and a higher risk of dysrhythmias in conditions with elevated IL6 levels. View Publication

BackgroundAniridia is a rare panocular disease caused by gene mutation in the PAX6,which is essential for eye development. Aniridia is inherited in an autosomal dominant manner,but its phenotype can vary significantly among individuals with the same mutation. Animal models,such as drosophila,zebrafish,and rodents,have been used to study aniridia through Pax6 deletions. Recently,patient-derived limbal epithelial stem cells (LESCs) and human-induced pluripotent stem cells (hiPSCs) have been used to model the disease in vitro,providing new insights into therapeutic strategies.MethodsIn this study,corneal organoids were generated from hiPSCs derived from aniridia patients with three different PAX6 nonsense mutations,allowing for a detailed comparison between diseased and healthy control models. These organoids structurally mimicked the human cornea and were used to investigate histologic and metabolomic differences between healthy and aniridia-derived samples.ResultsUntargeted metabolomic analysis revealed significant metabolic differences between wild-type (WT) and aniridia-associated keratopathy (AAK) hiPSCs. Further metabolomic profiling at different time points demonstrated distinct metabolic shifts,with amino acid metabolism pathways being consistently enriched in AAK organoids.ConclusionsThis study emphasizes the profound impact of AAK mutations on metabolism,particularly in amino acid biosynthesis and energy metabolism pathways.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12886-024-03831-w. View Publication

The blood?brain barrier (BBB) is a critical selective interface between the central nervous system (CNS) and the blood circulation. BBB dysfunction plays an important role in the neurological damage caused by sepsis. However,the mechanisms underlying the disruption of the BBB during sepsis remain unclear. We established a human induced pluripotent stem cell (iPSC)-derived BBB model and reported that treating with sepsis patient serum leads to structural and functional disruption of the BBB. In a cecal ligation and puncture (CLP)-induced mouse model of sepsis,we also observed disruption of the BBB,inflammation in the brain,and impairments in cognition. In both models,we found that the expression of TREM-1 was significantly increased in endothelial cells. TREM-1 knockout specifically in endothelial cells alleviated BBB dysfunction and cognitive impairments. Further study revealed that TREM-1 affects the expression of genes involved in the PI3K/Akt signaling pathway. The protective effects of TREM-1 inhibition on the BBB and cognition were abrogated by PI3K inhibitors. Our findings suggest that endothelial TREM-1 induces sepsis-induced BBB disruption and cognitive impairments via the PI3K/Akt signaling pathway. Targeting endothelial TREM-1 or the PI3K/Akt signaling pathway may be a promising strategy to maintain BBB integrity and improve cognitive function in sepsis patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03469-5. View Publication

Age-related macular degeneration (AMD),a multifactorial type of retinal degeneration represents the most common cause for blindness in elderly. Polymorphisms in complement factor-H increase,while absence of factor-H-related protein-1 (FHR1) decreases the AMD risk,currently explained by their opposing relationship. Here we identify a FHR1-driven pathway fostering chronic cellular inflammation. FHR1 accumulates below the retinal pigment epithelium (RPE) in AMD donor tissue and similarly the murine homolog,muFHR1 is abundant in three AMD-relevant mouse models. These mouse models express the muFHR1 receptor EGF-like module-containing mucin-like hormone receptor 1 (Emr1) on the RPE and on invading mononuclear phagocytes (MP),where both cells form clusters via muFHR1/Emr1. FHR1 ignited EMR2-dependent Ca2+-signals and gene expression in both human RPE cell line and in vivo where muFHR1 affects Emr1+ cells (RPE and MP) gene expression shown by RNAseq analysis. As muFHR1 deletion in mice revealed significantly reduced MP invasion and neoangiogenesis in laser-induced choroidal neovascularization,we hypothesize that FHR1 accumulates,stabilizes and activates MP in the stage of RPE degeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03499-z. View Publication

During the first month of pregnancy,the brain and spinal cord are formed through a process called neurulation. However,this process can be altered by low serum levels of folic acid,environmental factors,or genetic predispositions. In 2018,a surveillance study in Botswana,a country with a high incidence of human immunodeficiency virus (HIV) and lacking mandatory food folate fortification programs,found that newborns whose mothers were taking dolutegravir (DTG) during the first trimester of pregnancy had an increased risk of neural tube defects (NTDs). As a result,the World Health Organization and the U.S. Food and Drug Administration have issued guidelines emphasizing the potential risks associated with the use of DTG-based antiretroviral therapies during pregnancy. To elucidate the potential mechanisms underlying the DTG-induced NTDs,we sought to assess the potential neurotoxicity of DTG in stem cell-derived brain organoids. The gene expression of brain organoids developed in the presence of DTG was analyzed by RNA sequencing,Optical Coherence Tomography (OCT),Optical Coherence Elastography (OCE),and Brillouin microscopy. The sequencing data shows that DTG induces the expression of the folate receptor (FOLR1) and modifies the expression of genes required for neurogenesis. The Brillouin frequency shift observed at the surface of DTG-exposed brain organoids indicates an increase in superficial tissue stiffness. In contrast,reverberant OCE measurements indicate decreased organoid volumes and internal stiffness. View Publication

BackgroundHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling,drug discovery and potential therapeutic applications. However,conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA),which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally,the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored.MethodshiPSC-CMs were generated by Wnt pathway inhibition,followed by sodium l-lactate purification. Subsequently,biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar,sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function.ResultshiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%,which was significantly greater than DMSO (69.4?±?6.4%). A rapid cooling rate of 5 °C/min and a low nucleation temperature of -8 °C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw,dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw.ConclusionsIt was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04384-5. View Publication

With the advent of increasingly sophisticated organoids,there is growing demand for technology to replicate the interactions between multiple tissues or organs. This is challenging to achieve,however,due to the varying culture conditions of the different cell types that make up each tissue. Current methods often require complicated microfluidic setups,but fragile tissue samples tend not to fare well with rough handling. Furthermore,the more complicated the human system to be replicated,the more difficult the model becomes to operate. Here,we present the development of a multi-tissue chip platform that takes advantage of the modularity and convenient handling ability of a CUBE device. We first developed a blood-brain barrier-in-a-CUBE by layering astrocytes,pericytes,and brain microvascular endothelial cells in the CUBE,and confirmed the expression and function of important tight junction and transporter proteins in the blood-brain barrier model. Then,we demonstrated the application of integrating Tissue-in-a-CUBE with a chip in simulating the in vitro testing of the permeability of a drug through the blood-brain barrier to the brain and its effect on treating the glioblastoma brain cancer model. We anticipate that this platform can be adapted for use with organoids to build complex human systems in vitro by the combination of multiple simple CUBE units. Development of platform to integrate multiple Tissue-in-a-CUBEs in a chip for tissue-tissue interaction,demonstrated by simulating the testing of the permeability and effect of a cancer drug in a BBB-Brain cancer model. View Publication

Background/Objectives: High-altitude environments have a significant detrimental impact on the cognitive functions of the brain. Qi Jing Wan (QJW),a traditional herbal formula composed of Angelica sinensis,Astragalus membranaceus,and Rhizoma Polygonati Odorati,has demonstrated potential efficacy in treating cognitive disorders. However,its effects on cognitive dysfunction in plateau hypoxic environments remain unclear. Methods: In this study,acute and chronic plateau cognitive impairment mouse models were constructed to investigate the preventive and therapeutic effects of QJW and its significant active ingredient,diosgenin (Dio). Behavioral experiments were conducted to assess learning and memory in mice. Morphological changes in hippocampal neurons and synapses were assessed,and microglial activation and inflammatory factor levels were measured to evaluate brain damage. Potential active ingredients capable of crossing the blood–brain barrier were identified through chemical composition analysis and network database screening,followed by validation in animal and brain organoid experiments. Transcriptomics analysis,immunofluorescence staining,and molecular docking techniques were employed to explore the underlying mechanisms. Results: QJW significantly enhanced learning and memory abilities in plateau model mice,reduced structural damage to hippocampal neurons,restored NeuN expression,inhibited inflammatory factor levels and microglial activation,and improved hippocampal synaptic damage. Transcriptomics analysis revealed that Dio alleviated hypoxic brain damage and protected cognitive function by regulating the expression of PDE4C. Conclusions: These findings indicate that QJW and its significant active ingredient Dio effectively mitigate hypoxic brain injury and prevent cognitive impairment in high-altitude environments. View Publication

ABSTRACTRecent evidence suggests that atorvastatin exacerbates paclitaxel neurotoxicity via P?glycoprotein inhibition. We used a translational approach to investigate if atorvastatin or simvastatin exacerbates (i) paclitaxel neurotoxicity in human sensory neurons and (ii) paclitaxel?induced peripheral neuropathy (PIPN) in cancer patients. Paclitaxel neurotoxicity was assessed by quantifying neuronal networks of human induced pluripotent stem cell?derived sensory neurons (iPSC?SNs) with and without atorvastatin or simvastatin exposure. We estimated the odds ratio (OR) of early paclitaxel discontinuation due to PIPN in a nationwide cohort of paclitaxel?treated women (2014–2018),comparing atorvastatin users to simvastatin users and nonusers of statins. Only the highest concentration of atorvastatin (100?nM) significantly exacerbated paclitaxel neurotoxicity in iPSC?SNs (p? View Publication

The transmembrane death receptor Fas transduces apoptotic signals upon binding its ligand,FasL. Although Fas is highly expressed in cancer cells,insufficient cell surface Fas expression desensitizes cancer cells to Fas-induced apoptosis. Here,we show that the increase in Fas microaggregate formation on the plasma membrane in response to the inhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis. We used a clinically accessible Rho-kinase inhibitor,fasudil,that reduces endocytosis dynamics by increasing plasma membrane tension. In combination with exogenous soluble FasL (sFasL),fasudil promoted cancer cell apoptosis,but this collaborative effect was substantially weaker in nonmalignant cells. The combination of sFasL and fasudil prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft mouse model. Our results demonstrate that sFasL has strong potential for apoptosis-directed cancer therapy when Fas microaggregate formation is augmented by mechano-inhibition of endocytosis. View Publication

Cerebral organoids offer significant potential for neuroscience research as complex in vitro models that mimic human brain development. However,challenges related to their quality and reproducibility hinder their reliability. Discrepancies in morphology,size,cellular composition,and cytoarchitectural organization limit their applications,particularly in disease modeling,drug screening,and neurotoxicity testing. Critically,current methods for organoid characterization often lack standardization,restricting their broader applicability. To address the need for standardized quality assessment of cerebral organoids,we developed a Quality Control (QC) methodology for 60-day cortical organoids,evaluating five key criteria using a scoring system: morphology,size and growth profile,cellular composition,cytoarchitectural organization,and cytotoxicity. We implemented a hierarchical approach,beginning with non-invasive assessments to exclude low-quality organoids,while reserving in-depth analyses for those that passed the initial evaluation. To validate this framework,we exposed 60-day cortical organoids to graded doses of hydrogen peroxide (H2O2),inducing a range of quality outcomes. The QC system demonstrated its robustness by accurately discriminating organoid qualities. Our proposed QC framework is designed to be user-friendly,flexible,and broadly applicable,making it suitable for routine assessment of cerebral organoid quality. Additionally,its scalability enables industrial applications,offering a valuable tool for advancing both fundamental and pre-clinical research.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-14425-x. View Publication

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization,including disrupted nucleocytoplasmic transport (NCT); however,the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons,which is sufficient to cause NCT deficits,nuclear pore abnormalities,and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further,GDE2 deficits are evident in human neural cell models of ALS,which display erroneous Wnt activation that,when inhibited,increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease. Synopsis Nuclear exclusion of TDP-43 is observed in various pathologies,but the physiological mechanisms that ensure its nuclear localization are not well-known. This work shows that inhibition of persistent Wnt activation in neurons by GDE2 prevents TDP-43 nuclear exclusion. GDE2 inhibits canonical Wnt signaling in adult postmitotic neurons.Sustained activation of canonical Wnt signaling in neurons disrupts the nuclear pore complex,impairs nucleocytoplasmic transport,and results in TDP-43 nuclear exclusion.iPS neurons from patients with C9orf72 ALS show decreased GDE2 expression and increased activation of canonical Wnt signaling.Inhibition of Wnt activation mitigates TDP-43 dysfunction in C9orf72 iPS neurons. GDE2 maintains TDP-43 nuclear localization by inhibiting Wnt activation in neurons. View Publication