技术资料

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here,we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific,CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells,which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons,we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1?Mb,including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type,implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner. Scalable CRISPRa screening of cis-regulatory elements in non-cancer cell lines has proved challenging. Here,the authors describe a scalable,CRISPR activation screening framework to identify regulatory element-gene pairs in diverse cell types including cancer cells and neurons. View Publication

hiPSC-derived blood–brain barrier (BBB) models are valuable for pharmacological and physiological studies,yet their translational potential is limited due to insufficient cell phenotypes and the neglection of the complex environment of the BBB. This study evaluates the plasma compatibility with hiPSC-derived microvascular endothelial-like cells to enhance the translational potential of in vitro BBB models. Therefore,plasma samples (sodium/lithium heparin,citrate,EDTA) and serum from healthy donors were tested on hiPSC-derived microvascular endothelial-like cells at concentrations of 100%,75%,and 50%. After 24 h,cell viability parameters were assessed. The impact of heparin-anticoagulated plasmas was further evaluated regarding barrier function and endothelial phenotype of differentiated endothelial-like cells. Finally,sodium-heparin plasma was tested in an isogenic triple-culture BBB model with continuous TEER measurements for 72 h. Only the application of heparin-anticoagulated plasmas did not significantly alter viability parameters compared to medium. Furthermore,heparin plasmas improved barrier function without increasing cell density and induced a von Willebrand factor signal. Finally,continuous TEER measurements of the triple-culture model confirmed the positive impact of sodium-heparin plasma on barrier function. Consequently,heparin-anticoagulated plasmas were proven to be compatible with hiPSC-derived microvascular endothelial-like cells. Thereby,the translational potential of BBB models can be substantially improved in the future. View Publication

The translocator protein 18 kDa (TSPO) is a multifunctional outer mitochondrial membrane protein associated with various aspects of mitochondrial physiology and multiple roles in health and disease. Here,we aimed to analyse the role of TSPO in the regulation of mitochondrial and cellular functions in a human neuronal cell model. We used the CRISPR/Cas9 technology and generated TSPO knockout (KO) and control (CTRL) variants of human-induced pluripotent stem cells (hiPSCs). In a multimodal phenotyping approach,we investigated cellular and mitochondrial functions in neural progenitor cells (NPCs),astrocytes,and neurons differentiated from hiPSC CTRL and TSPO KO cell lines. Our analysis revealed reduced mitochondrial respiration and glycolysis,altered Ca2+ levels in the cytosol and mitochondrial matrix,a depolarised MMP,and increased levels of reactive oxygen species,as well as a reduced cell size. Notably,TSPO deficiency was accompanied by reduced expression of the voltage-dependent anion channel (VDAC). We also observed a reduced TSPO and VDAC expression in cells derived from patients suffering from major depressive disorder (MDD). Considering the modulatory function of TSPO and the similar functional phenotype of cells derived from patients with depression,we discuss a role of TSPO in the etiology or pathology of MDD. In summary,our findings indicate a general impairment of mitochondrial function in TSPO knockout (KO) cells. This deepens our insight into the intricate role of TSPO in a range of physiological and pathological processes. View Publication

BackgroundSpecific microglia responses are thought to contribute to the development and progression of neurodegenerative diseases,including Parkinson’s disease (PD). However,the phenotypic acquisition of microglial cells and their role during the underlying neuroinflammatory processes remain largely elusive. Here,according to the multiple-hit hypothesis,which stipulates that PD etiology is determined by a combination of genetics and various environmental risk factors,we investigate microglial transcriptional programs and morphological adaptations under PARK7/DJ-1 deficiency,a genetic cause of PD,during lipopolysaccharide (LPS)-induced inflammation.MethodsUsing a combination of single-cell RNA-sequencing,bulk RNA-sequencing,multicolor flow cytometry and immunofluorescence analyses,we comprehensively compared microglial cell phenotypic characteristics in PARK7/DJ-1 knock-out (KO) with wildtype littermate mice following 6- or 24-h intraperitoneal injection with LPS. For translational perspectives,we conducted corresponding analyses in human PARK7/DJ-1 mutant induced pluripotent stem cell (iPSC)-derived microglia and murine bone marrow-derived macrophages (BMDMs).ResultsBy excluding the contribution of other immune brain resident and peripheral cells,we show that microglia acutely isolated from PARK7/DJ-1 KO mice display a distinct phenotype,specially related to type II interferon and DNA damage response signaling,when compared with wildtype microglia,in response to LPS. We also detected discrete signatures in human PARK7/DJ-1 mutant iPSC-derived microglia and BMDMs from PARK7/DJ-1 KO mice. These specific transcriptional signatures were reflected at the morphological level,with microglia in LPS-treated PARK7/DJ-1 KO mice showing a less amoeboid cell shape compared to wildtype mice,both at 6 and 24 h after acute inflammation,as also observed in BMDMs.ConclusionsTaken together,our results show that,under inflammatory conditions,PARK7/DJ-1 deficiency skews microglia towards a distinct phenotype characterized by downregulation of genes involved in type II interferon signaling and a less prominent amoeboid morphology compared to wildtype microglia. These findings suggest that the underlying oxidative stress associated with the lack of PARK7/DJ-1 affects microglia neuroinflammatory responses,which may play a causative role in PD onset and progression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03164-x. View Publication

AbstractINTRODUCTIONAfrica,home to 1.4 billion people and the highest genetic diversity globally,harbors unique genetic variants crucial for understanding complex diseases like neurodegenerative disorders. However,African populations remain underrepresented in induced pluripotent stem cell (iPSC) collections,limiting the exploration of population?specific disease mechanisms and therapeutic discoveries.METHODSTo address this gap,we established an open?access African Somatic and Stem Cell Bank.RESULTSIn this initial phase,we generated 10 rigorously characterized iPSC lines from fibroblasts representing five Nigerian ethnic groups and both sexes. These lines underwent extensive profiling for pluripotency,genetic stability,differentiation potential,and Alzheimer's disease and Parkinson's disease risk variants. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR?associated protein 9 technology was used to introduce frontotemporal dementia?associated MAPT mutations (P301L and R406W).DISCUSSIONThis collection offers a renewable,genetically diverse resource to investigate disease pathogenicity in African populations,facilitating breakthroughs in neurodegenerative research,drug discovery,and regenerative medicine.Highlights We established an open?access African Somatic and Stem Cell Bank.10 induced pluripotent stem cell lines from five Nigerian ethnic groups were rigorously characterized.Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR?associated protein 9 technology was used to introduce frontotemporal dementia?causing MAPT mutations.The African Somatic and Stem Cell Bank is a renewable,genetically diverse resource for neurodegenerative research. View Publication

The development of patient-derived cardiac disease models has advanced rapidly due to the progress of human-induced pluripotent stem cell (hiPSC) technologies. Many protocols detail individual parts of the entire workflow,from handling hiPSCs and differentiating them into cardiomyocytes to live contraction imaging via widefield/phase-contrast and fluorescence microscopy. Here,we propose a streamlined protocol that guides users through hiPSC culture,differentiation,expansion,and functional imaging of hiPSC cardiomyocytes. First,hiPSC maintenance and handling procedures are outlined. Differentiation occurs over a two-week period,followed by selective expansion to increase the yield of hiPSC cardiomyocytes. Comprehensive characterization and quantification enable detailed contraction profiling of these cells. Designed to be low-cost,this protocol is suited for applications in drug discovery,screening,and clinical testing of patient-specific phenotypes. The addition of cardiomyocyte expansion and automated analysis distinguishes our protocol from current approaches. Key features • Protocol for the maintenance,differentiation,and expansion of hiPSC cardiomyocytes.• Detailed guidance for characterization and functional imaging of hiPSC cardiomyocytes.• Streamlined workflow integrating state-of-the-art protocols streamlined into a cost-effective approach.• A complete timeline from hiPSC culture to contraction imaging achievable in as little as three weeks. View Publication

Alcohol use disorder (AUD) is the most prevalent substance use disorder worldwide. Acamprosate and naltrexone are anti-craving drugs used in AUD pharmacotherapy. However,molecular mechanisms underlying their anti-craving effect remain unclear. This study utilized a patient-derived induced pluripotent stem cell (iPSC)-based model system and anti-craving drugs that are used to treat AUD as “molecular probes” to identify possible mechanisms associated with alcohol craving. We examined the pathophysiology of craving and anti-craving drugs by performing functional genomics studies using iPSC-derived astrocytes and next-generation sequencing. Specifically,RNA sequencing performed using peripheral blood mononuclear cells from AUD patients with extreme values for alcohol craving intensity prior to treatment showed that inflammation-related pathways were highly associated with alcohol cravings. We then performed a genome-wide assessment of chromatin accessibility and gene expression profiles of induced iPSC-derived astrocytes in response to ethanol or anti-craving drugs. Those experiments identified drug-dependent epigenomic signatures,with IRF3 as the most significantly enriched motif in chromatin accessible regions. Furthermore,the activation of IRF3 was associated with ethanol-induced endoplasmic reticulum (ER) stress which could be attenuated by anti-craving drugs,suggesting that ER stress attenuation might be a target for anti-craving agents. In conclusion,we found that craving intensity was associated with alcohol consumption and treatment outcomes. Our functional genomic studies suggest possible relationships among craving,ER stress,IRF3 and the actions of anti-craving drugs. 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

Statin drugs lower blood cholesterol levels for cardiovascular disease prevention. Women are more likely than men to experience adverse statin effects,particularly new-onset diabetes (NOD) and muscle weakness. Here we find that impaired glucose homeostasis and muscle weakness in statin-treated female mice are associated with reduced levels of the omega-3 fatty acid,docosahexaenoic acid (DHA),impaired redox tone,and reduced mitochondrial respiration. Statin adverse effects are prevented in females by administering fish oil as a source of DHA,by reducing dosage of the X chromosome or the Kdm5c gene,which escapes X chromosome inactivation and is normally expressed at higher levels in females than males. As seen in female mice,we find that women experience more severe reductions than men in DHA levels after statin administration,and that DHA levels are inversely correlated with glucose levels. Furthermore,induced pluripotent stem cells from women who developed NOD exhibit impaired mitochondrial function when treated with statin,whereas cells from men do not. These studies identify X chromosome dosage as a genetic risk factor for statin adverse effects and suggest DHA supplementation as a preventive co-therapy. Women are more likely than men to experience adverse statin effects,particularly new-onset diabetes and muscle weakness. Here the authors show that statin-treated female mice show reduced levels of docosahexaenoic acid (DHA) and that the adverse effects are prevented by administering fish oil as a source of DHA,by reducing dosage of the X chromosome or the Kdm5c gene. View Publication

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor,Y-27632 can enhance hPSC survival and proliferation at high seeding density,dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result,colonies tend to contain heterogeneous mixtures of both modified and unmodified cells,making it difficult to isolate the desired clone buried within the colony. In this study,we report improved clonal expansion of hPSCs using a retinoic acid analogue,TTNPB. When combined with Y-27632,TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%),whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore,TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype,pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts,in part,by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion,TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications. A retinoic acid analogue,TTNPB,improves clonal expansion in adherent and suspension culture of hPSCs by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. 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

Presynaptic scaffold proteins,including liprin-?,RIM,and ELKS,are pivotal to the assembly of the active zone and regulating the coupling of calcium signals and neurotransmitter release,yet the underlying mechanism remains poorly understood. Here,we determined the crystal structure of the liprin-?2/RIM1 complex,revealing a multifaceted intermolecular interaction that drives the liprin-?/RIM assembly. Neurodevelopmental disease-associated mutations block the formation of the complex. Disrupting this interaction in cultured human neurons impairs synaptic transmission and reduces the readily releasable pool of synaptic vesicles. Super-resolution imaging analysis supports a role for liprin-? in recruiting RIM1 to the active zone,presumably by promoting the liquid–liquid phase separation (LLPS) of RIM1. Strikingly,the liprin-?/RIM interaction modulates the competitive distribution of ELKS1 and voltage-gated Ca2+ channels (VGCCs) in RIM1 condensates. Disrupting the liprin-?/RIM interaction significantly decreased VGCC accumulation in the condensed phase and rendered release more sensitive to the slow calcium buffer EGTA,suggesting an increased physical distance between VGCC and vesicular calcium sensors. Together,our findings provide a plausible mechanism of the liprin-?/RIM complex in regulating the coupling of calcium channels and primed synaptic vesicles via LLPS for efficient synaptic transmission and uncover the pathological implication of liprin-? mutations in neurodevelopmental disorders. Scaffolding proteins regulate the assembly of the active zone in presynaptic terminals,but the mechanisms of assembly remain poorly understood. This study solves the crystal structure of the liprin-?2/RIM1 complex and shows that this interaction is essential for synaptic transmission and the coupling of calcium channels with primed synaptic vesicles in an LLPS-dependent manner. View Publication