技术资料

The inner ear organs responsible for hearing (cochlea) and balance (vestibular system) are susceptible to oxidative stress due to the high metabolic demands of their sensorineural cells. Oxidative stress-induced damage to these cells can cause hearing loss or vestibular dysfunction,yet the precise mechanisms remain unclear due to the limitations of animal models and challenges of obtaining living human inner ear tissue. Therefore,we developed an in vitro oxidative stress model of the pre-natal human inner ear using otic progenitor cells (OPCs) derived from human-induced pluripotent stem cells (hiPSCs). OPCs,hiPSCs,and HeLa cells were exposed to hydrogen peroxide or ototoxic drugs (gentamicin and cisplatin) that induce oxidative stress to evaluate subsequent cell viability,cell death,reactive oxygen species (ROS) production,mitochondrial activity,and apoptosis (caspase 3/7 activity). Dose-dependent reductions in OPC cell viability were observed post-exposure,demonstrating their vulnerability to oxidative stress. Notably,gentamicin exposure induced ROS production and cell death in OPCs,but not hiPSCs or HeLa cells. This OPC-based human model effectively simulates oxidative stress conditions in the human inner ear and may be useful for modeling the impact of ototoxicity during early pregnancy or evaluating therapies to prevent cytotoxicity. 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

SummaryToxic signaling by extrasynaptic NMDA receptors (eNMDARs) is considered an important promoter of amyotrophic lateral sclerosis (ALS) disease progression. To exploit this therapeutically,we take advantage of TwinF interface (TI) inhibition,a pharmacological principle that,contrary to classical NMDAR pharmacology,allows selective elimination of eNMDAR-mediated toxicity via disruption of the NMDAR/TRPM4 death signaling complex while sparing the vital physiological functions of synaptic NMDARs. Post-disease onset treatment of the SOD1G93A ALS mouse model with FP802,a modified TI inhibitor with a safe pharmacology profile,stops the progressive loss of motor neurons in the spinal cord,resulting in a reduction in the serum biomarker neurofilament light chain,improved motor performance,and an extension of life expectancy. FP802 also effectively blocks NMDA-induced death of neurons in ALS patient-derived forebrain organoids. These results establish eNMDAR toxicity as a key player in ALS pathogenesis. TI inhibitors may provide an effective treatment option for ALS patients. Graphical abstract Highlights•eNMDARs promote ALS disease progression via the NMDAR/TRPM4 death signaling complex•TwinF interface inhibitor FP802 disrupts the NMDAR/TRPM4 death signaling complex•FP802 is therapeutically effective in an ALS mouse model•FP802 protects against NMDA-induced death in brain organoids from ALS patient iPSCs Yan et al. find that FP802,which provides neuroprotection by detoxifying eNMDARs through disruption of the NMDAR/TRPM4 complex,halts motor neuron loss in an ALS mouse model,reduces serum NfL levels,improves motor performance,and extends life expectancy. FP802 is also neuroprotective in brain organoids derived from ALS patients. View Publication

Human induced pluripotent stem cell-derived sensory neuron (iPSC-dSN) models are a valuable resource for the study of neurotoxicity but are affected by poor replicability and reproducibility,often due to a lack of optimization. Here,we identify experimental factors related to culture conditions that substantially impact cellular drug response in vitro and determine optimal conditions for improved replicability and reproducibility. Treatment duration and cell seeding density were both found to be significant factors,while cell line differences also contributed to variation. A replicable dose–response in viability was demonstrated after 48-h exposure to docetaxel or paclitaxel. Additionally,a replicable dose-dependent reduction in neurite outgrowth was demonstrated,demonstrating the applicability of the model for the examination of additional phenotypes. Overall,we have established an optimized iPSC-dSN model for the study of taxane-induced neurotoxicity. View Publication

Introduction: Pathogenic variants in canonical transforming growth factor β (TGFβ) signaling genes predispose patients to thoracic aortic aneurysm and dissection (TAAD),predominantly in aortic root. Although TAAD pathogenesis associated with TGFβ receptor defects is well characterized,distinct and redundant mechanisms of TGFβ isoforms in TAAD incidence and severity remain elusive. Objective: Here we examined the biological role of TGFB2 in smooth muscle cell (SMC) differentiation and investigated how TGFB2 defects can lead to regional TAAD manifestations. Methods: To characterize the role of TGFB2 in SMC differentiation and function,we employed human-induced pluripotent stem cell (hiPSC)-derived SMC differentiation,CRISPR/Cas9 gene editing,three-dimensional SMC constructs,and human aortic tissue samples. Results: Despite the similar effects of different TGFβ isoforms on hiPSC-derived SMC differentiation,siRNA experiments revealed that TGFB2 distinctively displays TGFBR3 dependence for signal transduction,an understudied TGFβ receptor in TAAD. Molecular evaluation of different thoracic aorta regions suggested TGFB2 and TGFBR3 enrichment in the aortic root tunica media. TGFB2 haploinsufficiency (TGFB2KO/+) and TGFB2 neutralization impaired the differentiation of second heart field-derived SMCs. TGFBR3KO/KO prevented the molecular rescue of TGFB2KO/+ by TGFB2 supplementation indicating the involvement of TGFBR3 in TGFB2-mediated SMC differentiation. Lastly,a missense TGFB2 variant (TGFB2G276R/+) caused mechanical defects in SMC tissue ring constructs that were rescued by TGFB2 supplementation or genetic correction. Conclusion: Our data suggests the distinct regulation and action of TGFB2 in SMCs populating the aortic root,while redundant activities of TGFβ isoforms provide implications about the milder TAAD aggressiveness of pathogenic TGFB2 variants. View Publication

Induced pluripotent stem cells (iPSCs) can be generated from various adult cells,genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however,their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells,here,we aimed to generate human iPSCs overexpressing interferon-beta (IFNB,IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-?. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation,IFNB-iPSCs formed embryoid bodies and upregulated endoderm,mesoderm,and some ectoderm markers. However,an upregulation of key neuroectoderm markers,PAX6 and LHX2,was compromised. A negative effect of IFN-? on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-?. The study describes new IFN-?-producing iPSC lines suitable for the generation of various types of IFN-?-producing cells for future experimental and clinical applications,and it unravels an inhibitory effect of IFN-? on stem cell neuroectoderm differentiation. View Publication

Human embryonic stem cells (hESCs) serve as a valuable in vitro model for studying early human developmental processes due to their ability to differentiate into all three germ layers. Here,we present a comprehensive multi-omics dataset generated by differentiating hESCs into cardiomyocytes via the mesodermal lineage,collecting samples at 10 distinct time points. We measured mRNA levels by mRNA sequencing (mRNA-seq),translation levels by ribosome profiling (Ribo-seq),and protein levels by quantitative mass spectrometry-based proteomics. Technical validation confirmed high quality and reproducibility across all datasets,with strong correlations between replicates. This extensive dataset provides critical insights into the complex regulatory mechanisms of cardiomyocyte differentiation and serves as a valuable resource for the research community,aiding in the exploration of mammalian development and gene regulation. View Publication

SummaryFocusing on the early stages of Alzheimer’s disease (AD) holds great promise. However,the specific events in neural cells preceding AD onset remain elusive. To address this,we utilized human-induced pluripotent stem cells carrying APPswe mutation to explore the initial changes associated with AD progression. We observed enhanced neural activity and early neuronal differentiation in APPswe cerebral organoids cultured for one month. This phenomenon was also evident when neural progenitor cells (NPCs) were differentiated into neurons. Furthermore,transcriptomic analyses of NPCs and neurons confirmed altered expression of neurogenesis-related genes in APPswe NPCs. We also found that the upregulation of reactive oxygen species (ROS) is crucial for early neuronal differentiation in these cells. In addition,APPswe neurons remained immature after initial differentiation with increased susceptibility to toxicity,providing valuable insights into the premature exit from the neural progenitor state and the increased vulnerability of neural cells in AD. Graphical abstract Highlights•APPswe organoids show increased neural activity and early differentiation•Enhanced ROS levels are necessary but insufficient to accelerate differentiation•Transcriptome analysis of APPswe NPCs shows gene expression shift to differentiation•Premature neural cells with APPswe exhibit increased vulnerability to toxicity Molecular biology; Neuroscience; Cell biology View Publication

Quantum dots,which won the Nobel Prize in Chemistry,have recently gained significant attention in precision medicine due to their unique properties,such as size-tunable emission,high photostability,efficient light absorption,and vibrant luminescence. Consequently,there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors,biomolecular imaging probes,and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs,which often require expensive and toxic precursors,as they offer several merits in eco-friendly synthesis,preparation from renewable sources,and cost-effective production. In this study,we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines,including human dermal fibroblasts,HeLa,cardiomyocytes,induced pluripotent stem cells,and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells,as they were taken up more by cells treated with tumor necrosis factor-? than untreated cells. In addition,our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents,which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases. View Publication

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach,we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However,both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore,signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo,we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically,BMP signaling is integrated by SOX2. The interpretation of the key developmental signal BMP remains poorly understood. Here,the authors show that the total time-integrated signaling controls differentiation in a stem cell embryo model and provide a possible mechanism. View Publication

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However,they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX),Daunorubicin (DNR),Epirubicin (EPI),and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class,or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this,we studied the effects of treatment of DOX,DNR,EPI,MTX,and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum,while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling,a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time,giving rise to four distinct gene expression response signatures,denoted as TOP2i early-acute,early-sustained,and late response genes,and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators,which mediate AC sensitivity across breast cancer patients. However,there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability,we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed,DOX response eQTLs are enriched in genes that respond to all TOP2i. Next,we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i,respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes,including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity. Author summaryAnthracycline drugs such as Doxorubicin are effective treatments for breast cancer; however,they can cause cardiotoxicity in some women. It is unclear whether women would experience the same toxicity for all drugs in this class,or whether specific drugs would be better tolerated in specific individuals. We used an in vitro system of induced pluripotent stem cell-derived cardiomyocytes from six healthy females to test the effects of five breast cancer drugs on cell heath and global gene expression. We identified a strong shared cellular and gene expression response to drugs from the same class. However,there is more variation in gene expression levels between individuals following treatment with each anthracycline compared to untreated cells. We found that many genes in regions previously associated with Doxorubicin-induced cardiotoxicity in cancer patients,respond to at least two drugs in the class. This suggests that drugs in the same class induce similar effects on an individual’s heart. This work contributes to our understanding of how drug response,in the context of off-target effects,varies across individuals. View Publication

Cell replacement therapy is a promising therapeutic option for dry age-related macular degeneration (AMD). In this study,we outline our design for scalable manufacture with appropriate quality gates and present in vivo data for establishing preclinical safety and efficacy of an induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) product,thus laying the foundation for Phase 1/2a trial approval in India (ClinicalTrials.gov ID: NCT06394232; date of registration: 23rd September 2024). Escalating doses of RPE cell suspension in immunocompromised animals demonstrated absence of tumor formation up to 9?months post-injection. Good Laboratory Practices (GLP) toxicology and tolerability studies in rabbits and non-human primates (NHP) respectively showed no major adverse events. RPE transplanted into immune suppressed RCS rats showed integration,neuroprotection and rescue of visual function. In addition,we provide a detailed description of the modifications in GMP manufacturing protocol to create a final product with a unique composition and Chemistry,Manufacturing and Controls (CMC) studies performed during product development. View Publication