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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

Inclusion body myositis (IBM) is an inflammatory myopathy that displays proximal and distal muscle weakness. At the histopathological level,the muscles of IBM patients show inflammatory infiltrates,rimmed vacuoles and mitochondrial changes. The etiology of IBM remains unknown,and there is a lack of validated disease models,biomarkers and effective treatments. To contribute to unveil disease underpins we developed a cell model based on myotubes derived from induced pluripotent stem cells (iPSC-myotubes) from IBM patients and compared the molecular phenotype vs. age and sex-paired controls (n?=?3 IBM and 4 CTL). We evaluated protein histological findings and the gene expression profile by mRNA-seq,alongside functional analysis of inflammation,degeneration and mitochondrial function. Briefly,IBM iPSC-myotubes replicated relevant muscle histopathology features of IBM,including aberrant expression of HLA,TDP-43 and COX markers. mRNA seq analysis identified 1007 differentially expressed genes (DEGs) (p-value adj? View Publication

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However,generation of human AID cell lines is challenging,especially in human embryonic stem cells (hESCs). Here,we develop HiHo-AID2,a streamlined procedure for rapid,one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines,including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03187-w. 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

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

Background: Takotsubo cardiomyopathy (TTC) is marked by an acute,transient,and reversible left ventricular systolic dysfunction triggered by stress,with endothelial dysfunction being one of its pathophysiological mechanisms. However,the precise molecular mechanism underlying the interaction between endothelial cells and cardiomyocytes during TTC remains unclear. This study reveals that exosomal miRNAs derived from endothelial cells exposed to catecholamine contribute to ion channel dysfunction in the setting of TTC. Methods: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were treated with epinephrine (Epi) or exosomes (Exo) from Epi-treated human cardiac microvascular endothelial cells (HCMECs) or Exo derived from HCMECs transfected with miR-126-3p. The immunofluorescence staining,flow cytometry,qPCR,single-cell contraction,intracellular calcium transients,patch-clamp,dual luciferase reporter assay and western blot were performed for the study. Results: Modeling TTC with high doses of epinephrine (Epi) treatment in hiPSC-CMs shows suppression of depolarization velocity (Vmax),prolongation of action potential duration (APD),and induction of arrhythmic events. Exo derived from HCMECs treated with Epi (Epi-exo) mimicked or enhanced the effects of Epi. Epi exposure led to elevated levels of miR-126-3p in both HCMECs and their exosomes. Exo enriched with miR-126-3p demonstrated similar effects as Epi-exo,establishing the crucial role of miR-126-3p in the mechanism of Epi-exo. Dual luciferase reporter assay coupled with gene mutation techniques identified that miR-126-3p was found to target the regulator of G-protein signaling 3 (RGS3) gene. Western blot and qPCR analyses confirmed that miR-126-3p-mimic reduced RGS3 expression in both HCMECs and hiPSC-CMs,indicating miR-126-3p inhibits RGS3 signaling. Additionally,miR-126-3p levels were significantly higher in the serum of TTC patients compared to healthy controls and patients who had recovered from TTC. Conclusions: Our study is the first to reveal that exosomal miR-126-3p,originating from endothelial cells,contributes to ion channel dysfunction by regulating RGS3 signaling in cardiomyocytes. These findings provide new perspectives on the pathogenesis of TTC and suggest potential therapeutic targets for treatment. View Publication

Background: Human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs),can undergo erythroid differentiation,offering a potentially invaluable resource for generating large quantities of erythroid cells. However,the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors,with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. Methods: In this study,we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies,including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs,morphological analysis,quantitative real-time PCR (qRT-PCR),western blotting,flow cytometry,karyotyping,cytospin,Benzidine-Giemsa staining,immunofluorescence assay,and high-speed cell imaging analysis,were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation,as well as their differentiated erythroid cells. Results: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells,whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. Conclusions: VIM disappear during the normal maturation of erythroid cells,whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform,we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization. View Publication

Cell signaling plays a critical role in neurodevelopment,regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing,scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq,an integrated approach that aims to quantify cytoplasmic and nuclear proteins,including those with post-translational modifications,and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells,alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines,induced pluripotent stem cells,and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories,shed light on gene regulatory relationships,and help explore the causes and effects of diverse cell signaling in neurodevelopment. Here,the authors demonstrate Phospho-seq,a single-cell multiomics method capable of quantifying chromatin accessibility alongside intracellular proteins,including post-translationally modified proteins. Then,they apply Phospho-seq to organoid models of neurodevelopment. View Publication

Cardiac differentiation of human induced pluripotent stem cells is readily achievable,yet derivation of mature cardiomyocytes has been a recognized limitation. Here,a mesoderm priming approach was engineered to boost the maturation of cardiomyocyte progeny derived from pluripotent stem cells under standard cardiac differentiation conditions. Functional and structural hallmarks of maturity were assessed through multiparametric evaluation of cardiomyocytes derived from induced pluripotent stem cells following transfection of the mesoderm transcription factor Brachyury prior to initiation of lineage differentiation. Transfection with Brachyury resulted in earlier induction of a cardiopoietic state as hallmarked by early upregulation of the cardiac-specific transcription factors NKX2.5,GATA4,TBX20. Enhanced sarcomere maturity following Brachyury conditioning was documented by an increase in the proportion of cells expressing the ventricular isoform of myosin light chain and an increase in sarcomere length. Mesoderm primed cells displayed increased reliance on mitochondrial respiration as determined by increased mitochondrial size and a greater basal oxygen consumption rate. Further,Brachyury priming drove maturation of calcium handling enabling transfected cells to maintain calcium transient morphology at higher external field stimulation rates and augmented both calcium release and sequestration kinetics. In addition,transfected cells displayed a more mature action potential morphology with increased depolarization and repolarization kinetics. Derived cells transfected with Brachyury demonstrated increased toxicity response to doxorubicin as determined by a compromise in calcium transient morphology. Thus,Brachyury pre-treatment here achieved a streamlined strategy to promote maturity of human pluripotent stem cell-derived cardiomyocytes establishing a generalizable platform ready for deployment. View Publication

BackgroundSmall extracellular vesicles (EVs),exemplified by exosomes,mediate intercellular communication by transporting proteins,mRNAs,and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However,whether palmitoylation regulates small EV secretion,remains largely unexplored.MethodsVacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated,identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1,while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication,SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo,mouse model was established by intraperitoneal injection with palmitoylation inhibitor,2-bromopalmitate (2BP) for 3 months.ResultsVMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically,palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore,inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX,an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication,we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance,increased apoptosis,and decreased proliferation rate of SSCLCs. In vivo,intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes,which were closely associated with the level of spermatogenic cell apoptosis and proliferation.ConclusionsOur study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells,and demonstrated its pivotal role in intercellular communication and SSC niche.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01529-6. View Publication

In this study,we explore the therapeutic feasibility of globotriaosylceramide (Gb3) synthase (A4GALT)-specific siRNA-loaded polyhistidine (pHis)-incorporated lipid nanoparticles (HLNPs) for Fabry disease (FD). HLNPs were developed to deliver siRNAs targeting A4GALT using a microfluidic device,with pHis aiding in endosome escape. The therapy was tested on GLA-knockout human-induced pluripotent-stem-cell-derived endothelial cells (GLA-KO-hiPSC-ECs) and podocytes (GLA-KO-hiPSC-PCs). GLA-KO-hiPSCs-ECs or -PCs,upon differentiation,were treated with A4GALT-siRNA-HLNP. Successful intracellular uptake of A4GALT-siRNA-HLNP was confirmed through fluorescence and electron microscopy in both cell types. A4GALT-siRNA-HLNP treatment confirmed both cell types’ stability at 5 ?g/mL. Increased Gb3 deposition and zebra body formation were detected in both cell types,but A4GALT-siRNA-HLNP treatment attenuated these FD phenotypes,demonstrating reduced expression of A4GALT through western blot analysis. RNA sequencing analysis revealed that the expression of transcripts associated with FD was restored by A4GALT-siRNA-HLNP treatment in GLA-KO-hiPSCs-ECs,whereas in GLA-KO-hiPSCs-PCs,this effect was relatively less pronounced. Suppression of A4GALT via siRNA/HLNP treatment significantly rescued FD phenotypes especially in EC,presenting a novel therapeutic approach for FD. Graphical abstract This study highlights the therapeutic potential of A4GALT-siRNA delivered via HLNPs for Fabry disease (FD). In GLA-KO-hiPSC-derived endothelial cells and podocytes,treatment reduced Gb3 accumulation,restored transcriptomic changes,and mitigated FD phenotypes,with stronger effects in endothelial cells,supporting its promise as a novel FD therapy. View Publication