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Inflammatory cytokines,particularly interferon-γ (IFN-γ),are markedly elevated in the peripheral blood of patients with immune checkpoint inhibitor-induced myocarditis (ICI-M). Endomyocardial biopsies from these patients also show GBP-associated inflammasome overexpression. While both factors are implicated in ICI-M pathophysiology,their interplay and cellular targets remain poorly characterized. Our aim was to elucidate how ICI-M-associated cytokines affect the viability and inflammatory responses of endothelial cells (ECs) and cardiomyocytes (CMs) using human induced pluripotent stem cell (hiPSC)-derived models. ECs and CMs were differentiated from the same hiPSC line derived from a healthy donor. Cells were exposed either to IFN-γ alone or to an inflammatory cytokine cocktail (CCL5,GZMB,IL-1β,IL-2,IL-6,IFN-γ,TNF-α). We assessed large-scale transcriptomic changes via microarray and evaluated inflammatory,apoptotic,and cell death pathways at cellular and molecular levels. hiPSC-ECs were highly sensitive to cytokine exposure,displaying significant mortality and marked transcriptomic changes in immunity- and inflammation-related pathways. In contrast,hiPSC-CM showed limited transcriptional changes and reduced susceptibility to cytokine-induced death. In both cell types,cytokine treatment upregulated key components of the inflammasome pathway,including regulators (GBP5,GBP6,P2X7,NLRC5),a core component (AIM2),and the effector GSDMD. Increased GBP5 expression and CASP-1 cleavage mirrored the findings found elsewhere in endomyocardial biopsies from ICI-M patients. This hiPSC-based model reveals a distinct cellular sensitivity to ICI-M-related inflammation,with endothelial cells showing heightened vulnerability. These results reposition endothelial dysfunction,rather than cardiomyocyte injury alone,as a central mechanism in ICI-induced myocarditis. Modulating endothelial inflammasome activation,particularly via AIM2 inhibition,could offer a novel strategy to mitigate cardiac toxicity while preserving antitumor efficacy. View Publication

Artificial skin models have emerged as valuable tools for evaluating cosmetic ingredients and developing treatments for skin regeneration. Among them,3D skin equivalent models (SKEs) using human primary skin cells are widely utilized and supported by standardized testing guidelines. However,primary cells face limitations such as restricted donor availability and challenges in conducting genotype-specific studies. To overcome these issues,recent approaches have focused on differentiating skin cells from human-induced pluripotent stem cells (hiPSCs). In this study,we developed a protocol to differentiate high-purity skin cells,such as fibroblasts (hFIBROs) and keratinocytes (hKERAs),from hiPSCs. To construct the hiPSC-derived SKE (hiPSC-SKE),a dermis was first formed by culturing a collagen and hFIBROs mixture within an insert. Subsequently,hKERAs were seeded onto the dermis,and keratinization was induced under air-liquid culture conditions to establish an epidermis. Histological analysis with hematoxylin and eosin staining confirmed that the hiPSC-SKE recapitulated the layered architecture of native human skin and expressed appropriate epidermal and dermal markers. Moreover,exposure to Triton X-100,a known skin irritant,led to marked epidermal damage and significantly reduced cell viability,validating the model’s functional responsiveness. These findings indicate that the hiPSC-SKE model represents a promising alternative for various skin-related applications,including the replacement of animal testing. View Publication

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data,while reducing turnover time and costs,and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay,a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes,we sought to introduce standardization steps to the protocol,improving the overall robustness of the PluriLum assay,as well as a shortening of the assay protocol. First,we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm,robust differentiation can be anticipated. In terms of reproducibility,exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data,compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol,resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid,both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally,we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter,which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion,we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing. View Publication

The immune-modulatory effects of mesenchymal stromal cells (MSCs) are widely used to treat inflammatory disorders,with indoleamine 2,4-dioxygenase-1 (IDO-1) playing a pivotal role in suppressing stimulated T-cell proliferation. Taking that three-dimensional (3D) cultures enhance MSCs’ anti-inflammatory properties compared with two-dimensional (2D) cultures,the differentially expressed miRNAs were examined. Thus,we identified hsa-miR-4662a-5p (miR-4662a) as a key inducer of IDO-1 via its suppression of bridging integrator-1 (BIN-1),a negative regulator of the IDO-1 gene. The IDO-1-inducing potential of miR-4662a was conserved across primary MSCs from various donors and sources but exhibited variability. Notably,iPSC-derived MSCs (iMSCs) demonstrated superior IDO-1 induction and immune-modulatory efficacy compared with their donor-matched primary MSCs. Accordingly,iMSCs expressing miR-4662a (4662a/iMSC) exhibited stronger suppressive effects on T-cell proliferation and more potent suppressive effects on graft-versus-host disease (GVHD),improving survival rates and reducing tissue damage in the liver and gut. Our results point to the therapeutic potential of standardized,off-the-shelf 4662a/iMSC as a robust immune-modulating cell therapy for GVHD. 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

Background: Alzheimer's disease (AD) is characterized by progressive amyloid beta (Aβ) deposition in the brain,with eventual widespread neurodegeneration. While the cell-specific molecular signature of end-stage AD is reasonably well characterized through autopsy material,less is known about the molecular pathways in the human brain involved in the earliest exposure to Aβ. Human model systems that not only replicate the pathological features of AD but also the transcriptional landscape in neurons,astrocytes and microglia are crucial for understanding disease mechanisms and for identifying novel therapeutic targets. Methods: In this study,we used a human 3D iPSC-derived neurosphere model to explore how resident neurons,microglia and astrocytes and their interplay are modified by chronic amyloidosis induced over 3-5 weeks by supplementing media with synthetic Aβ1 - 42 oligomers. Neurospheres under chronic Aβ exposure were grown with or without microglia to investigate the functional roles of microglia. Neuronal activity and oxidative stress were monitored using genetically encoded indicators,including GCaMP6f and roGFP1,respectively. Single nuclei RNA sequencing (snRNA-seq) was performed to profile Aβ and microglia driven transcriptional changes in neurons and astrocytes,providing a comprehensive analysis of cellular responses. Results: Microglia efficiently phagocytosed Aβ inside neurospheres and significantly reduced neurotoxicity,mitigating amyloidosis-induced oxidative stress and neurodegeneration following different exposure times to Aβ. The neuroprotective effects conferred by the presence of microglia was associated with unique gene expression profiles in astrocytes and neurons,including several known AD-associated genes such as APOE. These findings reveal how microglia can directly alter the molecular landscape of AD. Conclusions: Our human 3D neurosphere culture system with chronic Aβ exposure reveals how microglia may be essential for the cellular and transcriptional responses in AD pathogenesis. Microglia are not only neuroprotective in neurospheres but also act as key drivers of Aβ-dependent APOE expression suggesting critical roles for microglia in regulating APOE in the AD brain. This novel,well characterized,functional in vitro platform offers unique opportunities to study the roles and responses of microglia to Aβ modelling key aspects of human AD. This tool will help identify new therapeutic targets,accelerating the transition from discovery to clinical applications. View Publication

ObjectiveThyroid hormone (TH) action is mediated by thyroid hormone receptor (THR) isoforms. While THR?1 is likely the main isoform expressed in liver,its role in human hepatocytes is not fully understood.MethodsTo elucidate the role of THR?1 action in human hepatocytes we used CRISPR/Cas9 editing to knock out THR?1 in induced pluripotent stem cells (iPSC). Following directed differentiation to the hepatic lineage,iPSC-derived hepatocytes were then interrogated to determine the role of THR?1 in ligand-independent and -dependent functions.ResultsWe found that the loss of THR?1 promoted alterations in proliferation rate and metabolic pathways regulated by T3,including gluconeogenesis,lipid oxidation,fatty acid synthesis,and fatty acid uptake. We observed that key genes involved in liver metabolism are regulated through both T3 ligand-dependent and -independent THR?1 signaling mechanisms. Finally,we demonstrate that following THR?1 knockout,several key metabolic genes remain T3 responsive suggesting they are THR? targets.ConclusionsThese results highlight that iPSC-derived hepatocytes are an effective platform to study mechanisms regulating TH signaling in human hepatocytes. Graphical abstractImage 1 Highlights•THR?1 is essential for T3 effects in human iPSC-derived hepatocytes (iHEPs).•THR?1 knockout reduces iPSC and progenitor cell proliferative capacity.•T3 regulates key genes involved in lipid and carbohydrate metabolism through THR?1.•THR?1 plays a strong ligand-independent role. View Publication

IntroductionEmerging biotechnologies are increasingly being explored for food production,including the development of cell-cultivated meat. Conventional approaches typically rely on satellite cell (SC) biopsies,which present challenges in scalability. Bovine induced pluripotent stem cells (biPSCs) represent a promising alternative due to their capacity for self-renewal and developmental plasticity.MethodsThis study utilized both lentiviral (integrating) and episomal (non-integrating) reprogramming strategies to generate biPSCs suitable for myogenic differentiation. Bovine fetal fibroblasts (bFFs) were reprogrammed using episomal vectors pMaster K and pCXB-EBNA1,leading to the emergence of putative iPSC colonies 13 days post-nucleofection. A clonal line,bFF-iPSCs pMK,was selected for further analysis.ResultsThe bFF-iPSCs pMK line expressed key pluripotency markers including alkaline phosphatase (AP),OCT4,SOX2,and NANOG,and was stably maintained for over 33 passages,although episomal plasmids remained detectable. in vitro myogenic differentiation was assessed by comparing this line to a previously established lentiviral reprogrammed line (bFF-iPSCs mOSKM). Both lines exhibited downregulation of pluripotency markers and upregulation of the early myogenic marker PAX3. By day 30,the bFF-iPSCs pMK line formed elongated,multinucleated cells characteristic of myotubes and displayed a corresponding gene expression profile.DiscussionThese results provide new insights into bovine in vitro myogenesis and its application in cultured meat production. While promising,the study also highlights the difficulty in achieving complete myogenic differentiation,indicating a need for further optimization of differentiation protocols. Graphical abstract View Publication

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure–function. This includes studies centered on disease etiology,drug development,and for potential clinical applications in heart regeneration/repair. Ultimately,for these applications to achieve success,a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state,and this represents a major hurdle for the field. To address this roadblock,we have developed a hiPSC-CMs (?-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere,the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile,the molecular motor of the heart,which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of ?-MyHC in ?-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis,we used gene editing with an inducible ?-MyHC expression cassette into isogeneic hiPSC-CMs,and separately by gene transfer,and then investigated the direct effects of increased ?-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with ?-MyHC. Positive inotropy and relaxation was evident in comparison to ?-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening,as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration,repair and re-muscularization applications. View Publication

BackgroundThree common isoforms of the apolipoprotein E (APOE) gene - APOE2,APOE3,and APOE4 - hold varying significance in Alzheimer’s Disease (AD) risk. The APOE4 allele is the strongest known genetic risk factor for late-onset Alzheimer’s Disease (AD),and its expression has been shown to correlate with increased central nervous system (CNS) amyloid deposition and accelerated neurodegeneration. Conversely,APOE2 is associated with reduced AD risk and lower CNS amyloid burden. Recent clinical data have suggested that increased blood-brain barrier (BBB) leakage is commonly observed among AD patients and APOE4 carriers. However,it remains unclear how different APOE isoforms may impact AD-related pathologies at the BBB.MethodsTo explore potential impacts of APOE genotypes on BBB properties and BBB interactions with amyloid beta,we differentiated isogenic human induced pluripotent stem cell (iPSC) lines with different APOE genotypes into both brain microvascular endothelial cell-like cells (BMEC-like cells) and brain pericyte-like cells. We then compared the effect of different APOE isoforms on BBB-related and AD-related phenotypes. Statistical significance was determined via ANOVA with Tukey’s post hoc testing as appropriate.ResultsIsogenic BMEC-like cells with different APOE genotypes had similar trans-endothelial electrical resistance,tight junction integrity and efflux transporter gene expression. However,recombinant APOE4 protein significantly impeded the “brain-to-blood” amyloid beta 1–40 (A?40) transport capabilities of BMEC-like cells,suggesting a role in diminished amyloid clearance. Conversely,APOE2 increased amyloid beta 1–42 (A?42) transport in the model. Furthermore,we demonstrated that APOE-mediated amyloid transport by BMEC-like cells is dependent on LRP1 and p-glycoprotein pathways,mirroring in vivo findings. Pericyte-like cells exhibited similar APOE secretion levels across genotypes,yet APOE4 pericyte-like cells showed heightened extracellular amyloid deposition,while APOE2 pericyte-like cells displayed the least amyloid deposition,an observation in line with vascular pathologies in AD patients.ConclusionsWhile APOE genotype did not directly impact general BMEC or pericyte properties,APOE4 exacerbated amyloid clearance and deposition at the model BBB. Conversely,APOE2 demonstrated a potentially protective role by increasing amyloid transport and decreasing deposition. Our findings highlight that iPSC-derived BBB models can potentially capture amyloid pathologies at the BBB,motivating further development of such in vitro models in AD modeling and drug development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-024-00580-2. 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

Background: Duchenne muscular dystrophy (DMD),which affects 1 in 3500 to 5000 newborn boys worldwide,is characterized by progressive skeletal muscle weakness and degeneration. The reduced muscle regeneration capacity presented by patients is associated with increased fibrosis. Satellite cells (SCs) are skeletal muscle stem cells that play an important role in adult muscle maintenance and regeneration. The absence or mutation of dystrophin in DMD is hypothesized to impair SC asymmetric division,leading to cell cycle arrest. Methods: To overcome the limited availability of biopsies from DMD patients,we used our 3D skeletal muscle organoid (SMO) system,which delivers a stable population of myogenic progenitors (MPs) in dormant,activated,and committed stages,to perform SMO cultures using three DMD patient-derived iPSC lines. Results: The results of scRNA-seq analysis of three DMD SMO cultures versus two healthy,non-isogenic,SMO cultures indicate reduced MP populations with constant activation and differentiation,trending toward embryonic and immature myotubes. Mapping our data onto the human myogenic reference atlas,together with primary SC scRNA-seq data,indicated a more immature developmental stage of DMD organoid-derived MPs. DMD fibro-adipogenic progenitors (FAPs) appear to be activated in SMOs. Conclusions: Our organoid system provides a promising model for studying muscular dystrophies in vitro,especially in the case of early developmental onset,and a methodology for overcoming the bottleneck of limited patient material for skeletal muscle disease modeling. View Publication