技术资料

The overproduction of reactive oxygen species (ROS) and mitochondrial dysregulation are regarded as key mechanisms in the progression of cardiac remodelling in cardiometabolic diseases including heart failure. Conventional treatments are often ineffective as they do not specifically target the underlying pathological mechanisms. Mitoquinone mesylate (MitoQ),a mitochondrial-targeted antioxidant has been reported to be protective against vascular dysfunction in hypertension,diabetic kidney disease and alcohol-induced liver damage. However,the cardioprotective potential of MitoQ to limit oxidative stress-induced mitochondrial remodelling in cardiomyocytes has not been fully resolved. We sought to investigate the effect of MitoQ and its mitochondrial-targeting moiety dodecyl-triphenylphosphonium (dTPP) on hydrogen peroxide-induced overproduction of ROS,mitochondrial dysregulation and cell death in H9C2 rat cardiomyoblasts (H9C2-rCM) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Cardiomyocytes were exposed to acute or chronic treatment (5–60 min or 48 h) of vehicle control (0.0001 % Ultrapure Milli-Q water),hydrogen peroxide (100 ?M) ± MitoQ (1 ?M) or dTPP (1 ?M) control. Hydrogen peroxide-induced overproduction of ROS,extracellular superoxide,mitochondrial ROS,mitochondrial hyperpolarisation and cell death were significantly blunted by MitoQ,but not dTPP,suggesting that the coenzyme Q10 moiety of MitoQ is protective under these conditions. Interestingly,both MitoQ and dTPP exhibited a pro-mitochondrial fusion effect by preserving mitochondrial network and reducing mitochondrial fragmentation in oxidative stress conditions. Overall,our findings confirm the cytoprotective potential of MitoQ to limit oxidative stress-induced adverse mitochondrial remodelling and dysregulation that is clinically observed in cardiometabolic-induced cardiac dysfunction in the failing heart. Graphical abstractBioRender Scientific Image and Illustration Software were used to generate the graphical abstract.Unlabelled Image Highlights•Oxidative stress is a key driver of mitochondrial dysregulation and cell death in cardiomyocytes.•MitoQ exhibits cytoprotection against elevated ROS production in human cardiomyocytes.•Mitochondrial structure and regulation were preserved in human cardiomyocytes with MitoQ treatment. View Publication

BackgroundThioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress,which disrupt glucose homeostasis in diabetes. However,the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear.MethodsWe used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP?/?) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP?/? and TXNIP+/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice.ResultsTXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency,viability,or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP?/? and TXNIP+/+ hESCs revealed differential expression of stress-responsive genes,with enriched apoptosis-related pathways in TXNIP+/+ cells,but minimal transcriptional changes specific to TXNIP deficiency. In HLCs,TXNIP deletion reduced albumin secretion and insulin signalling,as indicated by decreased AKT phosphorylation,while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress,TXNIP?/? HLCs exhibited transiently reduced eIF2? phosphorylation and lower BiP expression,suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP?/? hESCs showed comparable viability,endocrine cell composition,and cytokine responses to TXNIP+/+ islets. Following IFN? or IFN? treatment,STAT1 phosphorylation was increased in TXNIP?/? SC-islets,indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice,both TXNIP?/? and TXNIP+/+ SC-islets produced human C-peptide and responded to glucose stimulation. However,TXNIP?/? SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls.ConclusionsOur study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP?/? and TXNIP+/+ H1-hESCs,HLCs,and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04314-5. View Publication

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial roles in post-transcriptional gene regulation. Poly(A) RNA polymerase D5 (PAPD5) catalyzes the addition of adenosine to the 3? end of miRNAs. In this study,we demonstrate that the Yin Yang 1 protein,a transcriptional repressor of PAPD5,is recruited to both RNA foci and protein aggregates,resulting in an upregulation of PAPD5 expression in Huntington’s disease (HD). Additionally,we identify a subset of PAPD5-regulated miRNAs with increased adenylation and reduced expression in our disease model. We focus on miR-7-5p and find that its reduction causes the activation of the TAB2-mediated TAK1–MKK4–JNK pro-apoptotic pathway. This pathway is also activated in induced pluripotent stem cell-derived striatal neurons and post-mortem striatal tissues isolated from HD patients. In addition,we discover that a small molecule PAPD5 inhibitor,BCH001,can mitigate cell death and neurodegeneration in our disease models. This study highlights the importance of PAPD5-mediated miRNA dysfunction in HD pathogenesis and suggests a potential therapeutic direction for the disease. PAPD5 is responsible for adenylation of microRNAs. Here,the authors show that elevated level of PAPD5 enhances the adenylation and reduced expression of miR-7-5p. As a result,expression of TAB2,a target of miR-7-5p,is induced triggering neuronal apoptosis in Huntington’s disease. View Publication

Biallelic mutations in the DCPS gene disrupting the decapping activity of the scavenger decapping protein DcpS,leads to neurodevelopmental deficiencies and intellectual disability. However,the molecular basis for the neurogenesis defects in these individuals remains unknown. Here we show that cells derived from individuals with a DCPS mutation harbor a creatine deficiency and a corresponding elevation of the creatine precursor,guanidinoacetate (GAA). The altered metabolite levels are a consequence of a reduction in both the mRNA and protein levels for the enzyme that converts GAA into creatine,guanidinoacetate methyltransferase. Importantly,the compromised neurogenesis and neurite outgrowth phenotypes observed during the differentiation of DcpS mutant patient derived induced pluripotent stem cells into neurons was reversed upon supplementation of creatine monohydrate. These findings suggest creatine deficiency as an underlying factor for the neurogenetic defect detected in DcpS mutant cells and a potential driver of the neurological deficiencies in affected individuals. View Publication

Label-free detection of biological events at single-cell resolution in the brain can non-invasively capture brain status for medical diagnosis and basic neuroscience research. NADH is an universal coenzyme that not only plays a central role in cellular metabolism but may also be used as a biomarker to capture metabolic processes in brain cells and structures. We have developed a new label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H in living cells. The imaging depth of NAD(P)H in tissues with all-optical methods is limited to ~100??m in brain tissue by the strong absorption of the near-ultraviolet fluorescence. Here,acoustic detection of the thermal signature of multi-photon (three-photon) excitation of NAD(P)H,a low quantum yield fluorophore,allows detection at an unprecedented depth while the focused excitation ensures high spatial resolution. We validated the photoacoustic detection of NAD(P)H by monitoring an increase in intracellular NAD(P)H in HEK293T cells and HepG2 cells incubated in NADH solution. We also demonstrated the detection of endogenous NAD(P)H photoacoustic signals in brain slices to 700 ?m depth and in cerebral organoids to 1100 ?m depth. Finally,we developed and demonstrated simultaneous photoacoustic and optical imaging of NAD(P)H in brain cells with a real-time image acquisition and processing pipeline. This approach could open a new door to monitor brain metabolic changes during development and disease,and changes due to neuronal activity,at single-cell level deep in the brains of both humans and animals. Label-free,multiphoton photoacoustic microscope (LF-MP-PAM) with a near-infrared femtosecond laser to observe endogenous NAD(P)H of neurons in brain slices and cerebral organoids. View Publication

Angelman syndrome is a neurodevelopmental disorder caused by (epi)genetic lesions of maternal UBE3A. Research has focused largely on the role of UBE3A in neurons due to its imprinting in that cell type. Yet,evidence suggests there may be broader neurodevelopmental impacts of UBE3A dysregulation. Human cerebral organoids might reveal these understudied aspects of UBE3A as they recapitulate diverse cell types of the developing human brain. In this study,scRNAseq on organoids reveals the effects of UBE3A disruption on cell type-specific compositions and transcriptomic alterations. In the absence of UBE3A,progenitor proliferation and structures are disrupted while organoid composition shifts away from proliferative cell types. We observe impacts on non-neuronal cells,including choroid plexus enrichment. Furthermore,EMX1+ cortical progenitors are negatively impacted; potentially disrupting corticogenesis and delaying excitatory neuron maturation. This work reveals impacts of UBE3A on understudied cell types and related neurodevelopmental processes and elucidates potential therapeutic targets. Human cerebral organoids exhibit compositional and transcriptomic alterations in both neuronal and non-neuronal cells in the absence of UBE3A. View Publication

IntroductionNeuroinflammation is a key contributor to the pathogenesis of Alzheimer's disease (AD),and impaired clearance of amyloid-? (A?) by microglia is closely associated with disease progression. Oxytocin (OXT),a hypothalamic neuropeptide,has recently been reported to exert anti-inflammatory effects on microglia; however,its therapeutic potential in the human brain remains unclear.MethodsWe generated human cerebral organoids (hCOs) from induced pluripotent stem cells (iPSCs) to model early AD-like pathology. A? toxicity was induced by applying 3 ?M A?1–42 for 48 h. The protective effects of OXT were evaluated through immunohistochemistry,RT-qPCR,calcium imaging,and multielectrode array (MEA) recordings. The involvement of microglia in A? clearance was assessed by immunostaining and gene expression analysis of TREM2.ResultsA? exposure led to significant deposition of A? in the outer layers of hCOs,accompanied by suppressed neural activity and increased apoptotic signaling. Pretreatment with OXT attenuated A? deposition and caspase-3-mediated apoptosis in a concentration-dependent manner. OXT also restored calcium oscillations and neuronal network activity as measured by MEA. Notably,OXT enhanced the recruitment of microglia to A? deposits and upregulated the expression of TREM2,a key regulator of microglial phagocytosis. Co-expression of oxytocin receptors (OXTR) on Iba1-positive microglia suggests that OXT directly modulates microglial activation and A? clearance.ConclusionsOXT has neuroprotective effects on human cortical organoids by preserving their neuronal activity and promoting microglial-mediated A? clearance. This study provides novel insights into the therapeutic potential of OXT for targeting neuroinflammation and A? pathology in patients with AD. Graphical abstractImage 1 Highlights•Oxytocin reduces A? deposition and apoptosis in human cerebral organoids.•A? impairs neuronal activity,rescued by oxytocin preconditioning.•Oxytocin enhances microglial phagocytosis via OXTR and TREM2 upregulation.•Human iPSC-derived organoids model early A? pathology and oxytocin response. View Publication

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM),in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome,qRT-PCR,Metascape,and KEGG pathway analyses were conducted to assess elevated proteins,genes,and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-?),interleukin (IL)1-beta (IL-1?),IL-8 and IL-16. Enrichment analysis revealed malaria,positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria,MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally,HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ,activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore,our model is an alternative to rodent experimental models of CM. View Publication

Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study,we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor,based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway,ribosome-related,and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes,with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05622-y. View Publication

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes,but its efficacy is hindered by low oxygen supply post-transplantation,particularly in subcutaneous spaces and encapsulation devices,leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here,we show that ? cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes (EGR1,FOS,and JUN),which downregulates key ? cell transcription factors. We further identified genes important for maintaining ? cell fitness in hypoxia,with EDN3 as a potent player. Elevated EDN3 expression preserves ? cell identity and function in hypoxia by modulating genes involved in ? cell maturation,glucose sensing and regulation. These insights improve the understanding of hypoxia’s impact on stem cell-derived islets,offering a potential intervention for clinical applications. Hypoxia impairs the efficacy of stem cell-derived islet cell therapy,making it a potential barrier for treatment of type 1 diabetes. Wang et al. identify EDN3 as a key factor that preserves ? cell identity and function in hypoxia,offering possible strategies to improve therapeutic outcomes. View Publication

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here,to understand the pathological mechanisms of Kd,we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis,astrogenesis,and oligodendrogenesis but manifest early myelination defects. Specifically,Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly,myelin is affected in the absence of autophagy and mTOR pathway dysregulation,suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions,Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding,Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether,this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd. View Publication

Interactions between adipose tissue,liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model,we establish an interconnected microphysiological system (MPS) containing white adipocytes,hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS,we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function,whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance,corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases. In vitro modelling of the adipose tissue-liver axis can advance understanding and therapy of metabolic disease,including by distinguishing effects of obesity and inflammation. Here,authors develop such a system based on isogenic human iPSCs and interconnected microphysiological devices. View Publication