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

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

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

SummaryThyroid carcinoma represents the first malignancy among the endocrine organs. Investigating the cellular hierarchy and the mechanisms underlying the initiation of thyroid carcinoma is crucial in thyroid cancer research. Here,we present a protocol for deriving thyroid cell lineage from human embryonic stem cells. We also describe steps for engineering thyroid progenitor cells utilizing CRISPR-Cas9 technology,which can be used to perform in vivo studies,thus facilitating the development of representative thyroid tumorigenesis models.For complete details on the use and execution of this protocol,please refer to Veschi et al.1 Graphical abstract Highlights•Differentiation protocol for thyroid cell lineages from human embryonic stem cells•CRISPR-Cas9-mediated cellular engineering for common thyroid cancer genetic alteration•Orthotopic injection of thyroid progenitors to recapitulate thyroid cancer progression Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Thyroid carcinoma represents the first malignancy among the endocrine organs. Investigating the cellular hierarchy and the mechanisms underlying the initiation of thyroid carcinoma is crucial in thyroid cancer research. Here,we present a protocol for deriving thyroid cell lineage from human embryonic stem cells. We also describe steps for engineering thyroid progenitor cells utilizing CRISPR-Cas9 technology,which can be used to perform in vivo studies,thus facilitating the development of representative thyroid tumorigenesis models. View Publication

Visual Abstract Generation of human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) offers an unprecedented approach to modeling movement disorders such as dystonia and amyotrophic lateral sclerosis. However,achieving survival poses a significant challenge when culturing induced MNs,especially when aiming to reach late maturation stages. Utilizing hiPSC-derived motor neurons and primary mouse astrocytes,we assembled two types of coculture systems: direct coculturing of neurons with astrocytes and indirect coculture using culture inserts that physically separate neurons and astrocytes. Both systems significantly enhance neuron survival. Compared with these two systems,no significant differences in neurodevelopment,maturation,and survival within 3 weeks,allowing to prepare neurons at maturation stages. Using the indirect coculture system,we obtained highly pure MNs at the late mature stage from hiPSCs. Transcriptomic studies of hiPSC-derived MNs showed a typical neurodevelopmental switch in gene expression from the early immature stage to late maturation stages. Mature genes associated with neurodevelopment and synaptogenesis are highly enriched in MNs at late stages,demonstrating that these neurons achieve maturation. This study introduces a novel tool for the preparation of highly pure hiPSC-derived neurons,enabling the determination of neurological disease pathogenesis in neurons at late disease onset stages through biochemical approaches,which typically necessitate highly pure neurons. This advancement is particularly significant in modeling age-related neurodegeneration. View Publication

Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy,the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment,we observe that RING1 contributes to the broad placement of H2AK119ub1,and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1,reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely,reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast,hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression. Here,the authors establish a human in vitro model of neurodevelopment to investigate an allelic series of clinically relevant RING1 and RNF2 missense variants. The observations reveal that missense variants function according to a dominant-negative genetic mechanism. View Publication

BackgroundOrganoids,as near-physiological 3D culture systems,offer new opportunities to study the pathogenesis of various organs in mimicking the cellular complexity and functionality of human organs.MethodHere we used a quite simple and very practicable method to successfully generate induced pluripotent stem cell (iPSC)-derived human lung organoids (LuOrg) in a matrix-free manner as an alternative to the widely used preclinical mouse models in order to investigate normal lung damage in detail and as close as possible to the patient. We performed detailed morphological and molecular analyses,including bulk and single cell RNA sequencing,of generated lung organoids and evaluated the quality and robustness of our model as a potential in vitro platform for lung diseases,namely radiation-induced lung injury.ResultsA matrix-free method for differentiation of iPSCs can be used to obtain lung organoids that morphologically reflect the target tissue of the human lung very well,especially with regard to the cellular composition. The different cellular fates were investigated following the genotoxic stress induced by radiation and revealed further insights in the radiation-sensitivity of the different lung cells. Finally,we provide cellular gene sets found to be induced in the different lung organoid cellular subsets after irradiation,which could be used as additional RT response and particularly senescence gene sets in future studies.ConclusionBy establishing these free-floating LuOrgs for the investigation of cancer therapeutic approaches as a new and patient-oriented in vitro platform particularly in experimental radiooncology,not only a reduction in the number of experimental animals,but also an adequately and meaningfully replacement of corresponding animal experiments can be achieved.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-04106-3. Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-04106-3. View Publication

Human-induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) have been shown to be useful for the development of cell-based regenerative strategies and for modelling drug discovery. However,stem cell-derived HLCs are not identical in nature to primary human hepatocytes (PHHs),which could affect the cell phenotype and,potentially,model reliability. Therefore,we employed the in-depth gene expression profiling of HLCs and other important and relevant cell types,which led to the identification of clear similarities and differences between them at the transcriptional level. Through gene set enrichment analysis,we identified that genes that are critical for immune signalling pathways become downregulated upon HLC differentiation. Our analysis also found that TAV.HLCs exhibit a mild gene signature characteristic of acute lymphoblastic leukaemia,but not other selected cancers. Importantly,HLCs present significant similarity to PHHs,making them genuinely valuable for modelling human liver biology in vitro and for the development of prototype cell-based therapies for pre-clinical testing. View Publication