技术资料

Peripheral nerve and large-scale muscle injuries result in significant disability,necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials,graphene is favored for its high conductivity,but its low bioactivity requires enhancement through biomimetic components. In this study,we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds,aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia,observing significant improvements in axon outgrowth. Next,we tested the scaffolds with C2C12 mouse and human primary myoblasts,finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally,using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts,we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration,offering robust support for the growth and development of diverse neuromuscular tissues. View Publication

Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study,wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials,resulting in a unique band signal around 150 kDa for ADNP,above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting,comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal,indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector,we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore,we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations,while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies,suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However,western blotting of patient-derived hiPSCs,immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition,an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates,whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome. View Publication

Viruses depend on host metabolic pathways and flaviviruses are specifically linked to lipid metabolism. During dengue virus infection lipid droplets are degraded to fuel replication and Zika virus (ZIKV) infection depends on triglyceride biosynthesis. Here,we systematically investigated the neutral lipid–synthesizing enzymes diacylglycerol O-acyltransferases (DGAT) and the sterol O-acyltransferase (SOAT) 1 in orthoflavivirus infection. Downregulation of DGAT1 and SOAT1 compromises ZIKV infection in hepatoma cells but only SOAT1 and not DGAT inhibitor treatment reduces ZIKV infection. DGAT1 interacts with the ZIKV capsid protein,indicating that protein interaction might be required for ZIKV replication. Importantly,inhibition of SOAT1 severely impairs ZIKV infection in neural cell culture models and cerebral organoids. SOAT1 inhibitor treatment decreases extracellular viral RNA and E protein level and lowers the specific infectivity of virions,indicating that ZIKV morphogenesis is compromised,likely due to accumulation of free cholesterol. Our findings provide insights into the importance of cholesterol and cholesterol ester balance for efficient ZIKV replication and implicate SOAT1 as an antiviral target. Exploring the role of neutral lipid-synthesizing enzymes in Zika virus infection using different cell culture models,inhibition of cholesterol esterification is found to impair ZIKV morphogenesis. View Publication

ABSTRACTExtracellular vesicles (EVs) and secretory factors play crucial roles in intercellular communication,but the molecular mechanisms and dynamics governing their interplay in human pluripotent stem cells (hPSCs) are poorly understood. Here,we demonstrate that hPSC?secreted milk fat globule?EGF factor 8 (MFGE?8) is the principal corona protein at the periphery of EVs,playing an essential role in controlling hPSC stemness. MFGE?8 depletion reduced EV?mediated self?renewal and survival in hPSC cultures. MFGE?8 in the EV corona bound to integrin ?v?5 expressed in the peripheral zone of hPSC colonies. It activated cyclin D1 and dynamin?1 via the AKT/GSK3? axis,promoting the growth of hPSCs and facilitating the endocytosis of EVs. Internalization of EVs alleviated oxidative stress and cell death by transporting redox and stress response proteins that increased GSH levels. Our findings demonstrate the critical role of the extracellular association of MFGE?8 and EVs in modulating the self?renewal and survival of hPSCs. View Publication

To build in vitro tissues for therapeutic applications,it is essential to replicate the spatial distribution of cells that occurs during morphogenesis in vivo. However,it remains technically challenging to simultaneously regulate the geometric alignment and aggregation of cells during tissue fabrication. Here,we introduce the acoustofluidic bioassembly induced morphogenesis,which is the combination of precise arrangement of cells by the mechanical forces produced by acoustofluidic cues,and the morphological and functional changes of cells in the following in vitro and in vivo cultures. The acoustofluidic bioassembly can be used to create tissues with regulated nano-,micro-,and macro-structures. We demonstrate that the neuromuscular tissue fabricated with the acoustofluidic bioassembly exhibits enhanced contraction dynamics,electrophysiology,and therapeutic efficacy. The potential of the acoustofluidic bioassembly as an in situ application is demonstrated by fabricating artificial tissues at the defect sites of living tissues. The acoustofluidic bioassembly induced morphogenesis can provide a pioneering platform to fabricate tissues for biomedical applications. Tissue engineering is essential for drug screening and regenerative medicine. Here,authors developed an acoustofluidic method that can induce morphogenesis of therapeutic tissues at varied dimensions/scales. View Publication

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

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