技术资料

Friedreich ataxia (FRDA) is a multisystemic,autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently,there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice,and rescue was mediated by FXN transfer from tissue engrafted,HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation,we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease,such as non-homogenous microtubule staining in neurites,increased caspase-3 expression,mitochondrial superoxide levels,mitochondrial fragmentation,and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release,normalization of ER stress response gene,XBP-1,and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles,as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons,that was undetected in gene edited neurons. Taken together,these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA,validate the efficacy profile of our FXN gene editing approach in a disease relevant model,and support our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia. View Publication

IntroductionGlucose Transporter 1-Deficiency Syndrome (GLUT1-DS) is a rare genetic disorder caused by mutations in the gene encoding for GLUT1 and characterized by impaired glucose uptake in the brain. This leads to brain hypometabolism and the development of symptoms that include epilepsy,motor dysfunctions and cognitive impairment. The development of patient-specific in vitro models is a valuable tool for understanding the pathophysiology of rare genetic disorders and testing new therapeutic interventions.MethodsIn this study,we generated brain organoids from induced pluripotent stem cells (iPSCs) derived either from a GLUT1-DS patient or a healthy individual. The functional organoids were analyzed for cellular composition,maturity,and electrophysiological activity using a custom-made microelectrode array (MEA) platform,which allowed for the detection of spikes,burst patterns,and epileptiform discharges.ResultsImmunostaining revealed a similar distribution of neurons and astrocytes in both healthy and GLUT1-DS brain organoids,though GLUT1-DS brain organoids exhibited reduced cellular density and smaller overall size. Electrophysiological recordings demonstrated functional spike profiles in both organoid types. Notably,our study demonstrates that brain organoids derived from a GLUT1-DS patient exhibit distinct epileptiform activity and heightened sensitivity to glucose deprivation,reflecting key features of the disorder.DiscussionThese findings validate the use of brain organoids as a model for studying GLUT1-DS and highlight their potential for testing novel therapeutic strategies aimed at improving glucose metabolism and managing epilepsy in patients. View Publication

Recent studies demonstrate that brain infiltration of peripheral immune cells and their interaction with brain-resident cells contribute to Parkinson’s disease (PD). However,mechanisms of T cell-brain cell communication are not fully elucidated and models allowing investigation of interaction between T cells and brain-resident cells are required. In this study,we developed a three-dimensional (3D) model composed of stem cell-derived human midbrain organoids (hMO) and peripheral blood T cells. We demonstrated that organoids consist of multiple midbrain-specific cell types,allowing to study T cell motility and interactions with midbrain tissue in a spatially organized microenvironment. We optimized co-culture conditions and demonstrated that T cells infiltrate hMO tissue,leading to neural cell loss. Our work establishes a novel 3D cell co-culture model as a promising tool to investigate the effect of the adaptive immune system on the midbrain and can be used in future studies to address these processes in the context of PD. View Publication

Evolutionary innovations can be driven by changes in the rates of RNA translation and the emergence of new genes and small open reading frames (sORFs). In this study,we characterized the transcriptional and translational landscape of the hearts of four primate and two rodent species through integrative ribosome and transcriptomic profiling,including adult left ventricle tissues and induced pluripotent stem cell-derived cardiomyocyte cell cultures. We show here that the translational efficiencies of subunits of the mitochondrial oxidative phosphorylation chain complexes IV and V evolved rapidly across mammalian evolution. Moreover,we discovered hundreds of species-specific and lineage-specific genomic innovations that emerged during primate evolution in the heart,including 551 genes,504 sORFs and 76 evolutionarily conserved genes displaying human-specific cardiac-enriched expression. Overall,our work describes the evolutionary processes and mechanisms that have shaped cardiac transcription and translation in recent primate evolution and sheds light on how these can contribute to cardiac development and disease. Ruiz-Orera et al. used comparative transcriptomics and translatomics to analyze the cardiac evolution in primates and discovered species-specific and lineage-specific genomic innovations that might contribute to cardiac development and disease. View Publication

An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits,we investigated an in vitro neural tissue model for inter-regional connections,in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids,suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition,optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust short-term plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro. Connecting cerebral organoids with an axon bundle models inter-regional projections and enhances neural activity. Optogenetic stimulation induces short-term plasticity,offering insights into macroscopic circuit development and functionality. View Publication

Neutrophils are critical for host defense against fungi. However,the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here,we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation,antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal ?-glucan,including an upregulation of the pentose phosphate pathway (PPP),similar to primary human neutrophils in vitro. Furthermore,we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively,these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections,and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation,limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus,demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively,these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity. View Publication

SUMMARY During cell fate transitions,cells remodel their transcriptome,chromatin,and epigenome; however,it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here,we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that,following heterokaryon formation,the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG,conversely,has significant nascent RNA transcription only at 48 h after cell fusion but,strikingly,exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent. In brief Martinez-Sarmiento et al. demonstrate that,during heterokaryon reprogramming,global chromatin decondensation and loss of repressive histone modifications occur at late stages after cell fusion. Activation of OCT4 precedes global chromatin decompaction and does not require the opening of its local genomic region. Conversely,NANOG activation occurs after OCT4 activation,and the NANOG locus undergoes opening prior to its transcriptional activation. Graphical Abstract View Publication

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

Introduction: Africa,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. Methods: To address this gap,we established an open-access African Somatic and Stem Cell Bank. Results: In 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). Discussion: This 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