技术资料

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function,DYSF exon 44,founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing,rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation,hEx44mut,recapitulates the patients’ phenotype and an identical re-framing outcome in primary muscle stem cells. Finally,gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility,not only in treating dysferlin deficiency syndromes,but also perhaps other forms of muscular dystrophy. Dysferlin-deficient muscular dystrophy is a devastating and untreatable disease. Using Cas9,the authors restored dysferlin in muscle stem cells from patients ex vivo and show proof-of-concept for autologous cell replacement therapies in a new humanized mouse model. 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

The spatial organization of molecules in a cell is essential for their functions. While current methods focus on discerning tissue architecture,cell–cell interactions,and spatial expression patterns,they are limited to the multicellular scale. We present Bento,a Python toolkit that takes advantage of single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains,annotating localization patterns,and quantifying gene–gene colocalization. We demonstrate MERFISH,seqFISH +,Molecular Cartography,and Xenium datasets. Bento is part of the open-source Scverse ecosystem,enabling integration with other single-cell analysis tools.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03217-7. View Publication

SummaryCardiac fibrosis is a key characteristic of heart failure. CTPR390,an experimental anti-fibrotic inhibitor targeting Hsp90,has shown success in animal models,but remains unexplored in human cardiac models. This study evaluated an engineered cardiac connective tissue (ECCT) model,focusing on changes in the extracellular matrix and fibroblasts. Results showed that CTPR390 prevented architectural changes in TGF?1-activated ECCT,preserving tissue perimeter,collagen fibers alignment while reducing structured areas and degree of collagen structuration. CTPR390 treatment reduced cell area of fibroblasts under tension,without changes in the internal rounded cells devoid of tension. Fibroblast recruitment to tension areas was diminished,showing biomechanical behavior similar to control ECCT. This treatment also lowered the gene and protein expression of key pro-fibrotic markers. Here,advanced biotechnology was employed to detect the detailed structure of tissue fibrosis reduction after administering CTPR390,representing a significant advancement toward clinical application for cardiac fibrosis treatment. Graphical abstract Highlights•CTPR390 prevented architectural changes in TGF?1-activated ECCT•CTPR390 preserves tissue perimeter,collagen fibers alignment•CTPR390 reduces structured areas and degree of collagen structuration•CTPR390-trested ECCTs presented a biomechanical behavior similar to control ECCT Molecular biology; Cell biology 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

The blood?brain barrier (BBB) is a critical selective interface between the central nervous system (CNS) and the blood circulation. BBB dysfunction plays an important role in the neurological damage caused by sepsis. However,the mechanisms underlying the disruption of the BBB during sepsis remain unclear. We established a human induced pluripotent stem cell (iPSC)-derived BBB model and reported that treating with sepsis patient serum leads to structural and functional disruption of the BBB. In a cecal ligation and puncture (CLP)-induced mouse model of sepsis,we also observed disruption of the BBB,inflammation in the brain,and impairments in cognition. In both models,we found that the expression of TREM-1 was significantly increased in endothelial cells. TREM-1 knockout specifically in endothelial cells alleviated BBB dysfunction and cognitive impairments. Further study revealed that TREM-1 affects the expression of genes involved in the PI3K/Akt signaling pathway. The protective effects of TREM-1 inhibition on the BBB and cognition were abrogated by PI3K inhibitors. Our findings suggest that endothelial TREM-1 induces sepsis-induced BBB disruption and cognitive impairments via the PI3K/Akt signaling pathway. Targeting endothelial TREM-1 or the PI3K/Akt signaling pathway may be a promising strategy to maintain BBB integrity and improve cognitive function in sepsis patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03469-5. 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

The work highlights the different calcium channels involved in controlling protein synthesis in neurons,and shows the dysfunction of this process in Alzheimer’s disease neurons. Calcium signaling is integral for neuronal activity and synaptic plasticity. We demonstrate that the calcium response generated by different sources modulates neuronal activity–mediated protein synthesis,another process essential for synaptic plasticity. Stimulation of NMDARs generates a protein synthesis response involving three phases—increased translation inhibition,followed by a decrease in translation inhibition,and increased translation activation. We show that these phases are linked to NMDAR-mediated calcium response. Calcium influx through NMDARs elicits increased translation inhibition,which is necessary for the successive phases. Calcium through L-VGCCs acts as a switch from translation inhibition to the activation phase. NMDAR-mediated translation activation requires the contribution of L-VGCCs,RyRs,and SOCE. Furthermore,we show that IP3-mediated calcium release and SOCE are essential for mGluR-mediated translation up-regulation. Finally,we signify the relevance of our findings in the context of Alzheimer’s disease. Using neurons derived from human fAD iPSCs and transgenic AD mice,we demonstrate the dysregulation of NMDAR-mediated calcium and translation response. Our study highlights the complex interplay between calcium signaling and protein synthesis,and its implications in neurodegeneration. View Publication

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream,playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases,including amyotrophic lateral sclerosis (ALS); however,the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression,compromised barrier integrity,and increased P-glycoprotein transporter activity. In addition,mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover,our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally,C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-024-00528-6. 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

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study,we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs,this method prioritizes functional noncoding variants using deep learning,links cis-regulatory elements (CREs) to target genes using epigenomics data,and integrates these data through gene-level burden tests to identify survival-modifying variants,CREs,and genes. We apply this approach to analyze 6,715 ALS genomes,and pinpoint four novel rare noncoding variants associated with survival,including chr7:76,009,472:C>T linked to CCDC146. CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes,including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO),showing no toxicity,completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72. ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases. View Publication