技术资料

Spinal motor neurons (MNs) represent a highly vulnerable cellular population,which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study,we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/? hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover,analysis of the SYT13+/? transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures,displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes,which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family,SYT13,is sufficient to trigger a series of abnormal alterations leading to MN sufferance,thus revealing novel insights into the selective vulnerability of this cell population. View Publication

SUMMARY Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance,there are currently no stem-cell-derived models of human engineered cardiac tissues (hECTs) that include resident macrophages. In this study,we made an induced pluripotent stem cell (iPSC)-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. The inclusion of iM0s significantly impacted hECT function,increasing contractile force production. A potential mechanism underlying these changes was revealed by the interrogation of calcium signaling,which demonstrated the modulation of ?-adrenergic signaling in +iM0 hECTs. Collectively,these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models,emphasizing the need to further explore their contributions not only in healthy hECT models but also in the contexts of disease and injury. In brief Lock and Graney et al. develop a human engineered cardiac tissue with an incorporated iPSC-derived macrophage population to better mimic the complex cell landscape of the native myocardium. Macrophage inclusion leads to increased contractile function of the tissue,which is attributed to macrophage stimulation of the cardiomyocyte ?-adrenergic signaling pathway. Graphical Abstract View Publication

Characterization and modeling of biological neural networks has emerged as a field driving significant advancements in our understanding of brain function and related pathologies. As of today,pharmacological treatments for neurological disorders remain limited,pushing the exploration of promising alternative approaches such as electroceutics. Recent research in bioelectronics and neuromorphic engineering have fostered the development of the new generation of neuroprostheses for brain repair. However,achieving their full potential necessitates a deeper understanding of biohybrid interaction. In this study,we present a novel real-time,biomimetic,cost-effective and user-friendly neural network capable of real-time emulation for biohybrid experiments. Our system facilitates the investigation and replication of biophysically detailed neural network dynamics while prioritizing cost-efficiency,flexibility and ease of use. We showcase the feasibility of conducting biohybrid experiments using standard biophysical interfaces and a variety of biological cells as well as real-time emulation of diverse network configurations. We envision our system as a crucial step towards the development of neuromorphic-based neuroprostheses for bioelectrical therapeutics,enabling seamless communication with biological networks on a comparable timescale. Its embedded real-time functionality enhances practicality and accessibility,amplifying its potential for real-world applications in biohybrid experiments. Beaubois et al. introduce a real-time biomimetic neural network for biohybrid experiments,providing a tool to study closed-loop applications for neuroscience and neuromorphic-based neuroprostheses. View Publication

Montelukast (MTK) is a drug widely used for treating allergic rhinitis and asthma. However,severe neuropsychiatric adverse events related to MTK have been reported,with limited understanding of the underlying mechanisms. Here we leveraged human forebrain organoids (hFOs) and showed that MTK exposure in hFOs downregulated the expression of genes associated with multiple neuronal functions and neuropsychiatric disorders. The following integrative analysis highlighted adenylate cyclase 1 (ADCY1),a main regulator of the cAMP signaling pathway,as a hub gene mediating the functional effects of MTK exposure. We also showed that MTK exposure resulted in a reduction of cAMP and neuroactivities,and caused neural maturation defects. These cellular phenotypes could be recapitulated by treating hFOs with ST034307,a selective ADCY1 inhibitor,or partially rescued by ADCY1 overexpression in hFOs. Together,this study underscored that MTK exposure caused neuropsychiatric effects through inhibiting the ADCY1-mediated cAMP signaling pathway.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05764-z. View Publication

Becker muscular dystrophy (BMD) is caused by in-frame mutations in dystrophin gene,leading to progressive muscle weakness,and cardiac and respiratory complications. Currently,there is no cure. We have recently identified the importance of poly-ubiquitination in regulating dystrophin stability through the binding of lncRNA H19 to the dystrophin C-terminal zinc-finger domain (ZNF),inhibiting TRIM63-mediated poly-ubiquitination. We also demonstrated that BMD mutations lead to conformational changes in ZNF domain,reduced lncRNA H19 binding and increased dystrophin ubiquitination. Here we used BMD iPSCs to investigate the in vitro myogenic potential of BMD myogenic cells,as well as in vitro and in vivo studies to evaluate the therapeutic efficacy of three candidate molecules targeting dystrophin ubiquitination pathway. In vitro assays indicated significant deficiencies in myogenic cell differentiation of BMD iPSCs,including reduced proliferation,cell-cycle arrest,increased apoptosis,senescence,and membrane damage,and impaired myotube formation. In vivo engraftment demonstrated significant improvement in BMD iPSC myogenic cell survival and dystrophin expression in the animals treated with two molecules: a TRIM63 inhibitor and an ?-synuclein aggregation inhibitor. These findings provide promising evidence for the potential therapeutic efficacy of these ubiquitination pathway inhibitors to improve muscle progenitor cell survival and dystrophin expression in BMD patients. Graphical abstract Regulation of dystrophin stability via poly-ubiquitination is crucial in Becker muscular dystrophy (BMD). BMD mutations impair lncRNA H19 binding,increasing dystrophin ubiquitination. Darabi and colleagues’ studies,using BMD iPSCs and in vivo models,demonstrate that inhibiting TRIM63 or ?-synuclein aggregation improves myogenic cell survival and dystrophin expression,suggesting promising therapeutic avenues for BMD. View Publication

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking,which has impeded their broader application. In this study,we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics,immunogenicity,immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs),iMSCs demonstrated an enhanced proliferative capacity,a higher level of regenerative gene expression,and lower immunogenicity,strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition,iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models,iMSCs engrafted in the lungs,liver,and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity,low immunogenicity and unique immunomodulatory properties,and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects. View Publication

ABSTRACTDuchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder affecting 1:3500 male births and is associated with myofiber degeneration,regeneration,and inflammation. Glucocorticoid treatments have been the standard of care due to immunomodulatory/immunosuppressive properties but novel genetic approaches,including exon skipping and gene replacement therapy,are currently being developed. The identification of additional biomarkers to assess DMD-related inflammatory responses and the potential efficacy of these therapeutic approaches are thus of critical importance. The current study uses RNA sequencing of skeletal muscle from two mdx mouse models to identify high mobility group box 1 (HMGB1) as a candidate biomarker potentially contributing to DMD-related inflammation. HMGB1 protein content was increased in a human iPSC-derived skeletal myocyte model of DMD and microdystrophin treatment decreased HMGB1 back to control levels. In vivo,HMGB1 protein levels were increased in vehicle treated B10-mdx skeletal muscle compared to B10-WT and significantly decreased in B10-mdx animals treated with adeno-associated virus (AAV)-microdystrophin. However,HMGB1 protein levels were not increased in D2-mdx skeletal muscle compared to D2-WT,demonstrating a strain-specific difference in DMD-related immunopathology. Summary: Duchenne muscular dystrophy is a devastating that currently has limited treatment options. RNA sequencing and downstream analysis in iSkM and mdx samples revealed HMGB1 may be a relevant treatment biomarker. View Publication

Developmental and epileptic encephalopathies (DEEs) feature altered brain development,developmental delay and seizures,with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A,encoding a membrane trafficking protein,and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1,components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles,biasing daughter cells towards a more fate-committed state,ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families. Developmental and epileptic encephalopathies are devastating neurological disorders. Here,the authors establish a cohort of patients with variants in the gene DENND5A and use human stem cells to discover a disease mechanism involving altered cell division. View Publication

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. View Publication

Angelman syndrome (AS) and Prader-Willi syndrome (PWS),two distinct neurodevelopmental disorders,result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele,respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless,gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences,reprogramming methods,and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here,we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally,these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. View Publication

BackgroundHuman pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI),but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models,representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore,we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.MethodsWe used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs,and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next,we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.ResultsIn vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (If) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs,all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However,in contrast to our hypothesis,both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).ConclusionsWhile genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro,this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model,implying more complex mechanism(s) are operational in vivo. View Publication

Cardiac hypertrophy is a cellular process characterized by the increased size of cardiomyocytes in response to a high workload or stress. 17-beta estradiol (E2) has cardioprotective and anti-hypertrophic effects by maintaining mitochondrial network and function. MUL1 is a mitochondrial ubiquitin ligase directly involved in the control of mitochondrial fission and mitophagy. Studies from our group and others have previously shown that cardiomyocyte hypertrophy is associated with mitochondrial fission and dysfunction. These findings led us to study in vitro whether E2 regulates MUL1 to prevent cardiac hypertrophy,mitochondrial fission,and dysfunction induced by the catecholamine norepinephrine (NE). Our results showed that NE induces hypertrophy in cultured rat cardiomyocytes. Pre-treatment with E2 (10-100?nM) prevented the NE-dependent increases in cell perimeter and the hypertrophic stress markers ANP and BNP at both the protein and mRNA levels. NE induced the fragmentation of the mitochondrial network and reduced ATP levels,effects that were both prevented by E2. In silico analysis suggested a putative binding site for estrogen receptors on the MUL1 gene promoter. In accordance with this finding,E2 prevented increases in MUL1 mRNA and protein levels induced by NE. Our data also showed that a siRNA MUL1 knockdown counteracted NE-induced cardiomyocyte hypertrophy and mitochondrial dysfunction,mirroring the protective effect triggered by E2. In contrast,a MUL1 adenovirus did not prevent the E2 protection from cardiomyocyte hypertrophy. Further,in vivo analysis in a transgenic mouse model overexpressing MUL1 revealed that only young male mice overexpressed the protein. Consequently,they exhibited increased levels of the hypertrophic marker ANP,an elevated heart weight,and larger cardiomyocyte size. Therefore,our data demonstrate that 17-beta estradiol prevents cardiac myocyte hypertrophy by regulating MUL1. View Publication