参考文献

N6-methyladenosine (m6A) is an abundant internal RNA modification that can impact gene expression at both post-transcriptional and transcriptional levels. However,the landscapes and functions of m6A in human brains and neurodegenerative diseases,including Alzheimer’s disease (AD),are under-explored. Here,we examined RNA m6A methylome using total RNA-seq and meRIP-seq in middle frontal cortex of post-mortem brains from individuals with or without AD,which revealed m6A alteration on both mRNAs and various noncoding RNAs. Notably,many promoter-antisense RNAs (paRNAs) displayed cell-type-specific expression and changes in AD,including one produced adjacent to MAPT that encodes the Tau protein. MAPT-paRNA is highly expressed in neurons,and m6A positively controls its expression. In iPSC-derived human excitatory neurons,MAPT-paRNA does not impact the nearby MAPT mRNA,but instead promotes expression of hundreds of neuronal and synaptic genes,and is protective against excitotoxicity. Analysis of single nuclei RNA-DNA interactome in human brains supports that brain paRNAs interact with both cis- and trans-chromosomal target genes to impact their transcription. These data reveal landscapes and functions of noncoding RNAs and m6A in brain gene regulation and AD pathogenesis. This study characterised the landscapes and changes of RNA m6A in brains of individuals with or without Alzheimer’s disease,and revealed roles of a promoter antisense RNA next to MAPT in neuronal gene regulation that promote neuronal survival. View Publication

Biallelic mutations in PIGK cause GPI biosynthesis defect 22 (GPIBD22),characterized with developmental delay,hypotonia,and cerebellar atrophy. The understanding of the underlying causes is limited due to the lack of suitable disease models. To address this gap,we generated a mouse model with PIGK deficits,specifically in Purkinje cells (Pcp2-cko) and an induced pluripotent stem cell (iPSC) model using the c.87dupT mutant (KI) found in GPIBD22 patients. Pcp2-cko mice demonstrated cerebellar atrophy,ataxia and progressive Purkinje cells loss which were accompanied by increased apoptosis and neuroinflammation. Similarly,KI iPSCs exhibited increased apoptosis and accelerated neural rosette formation,indicating that PIGK defects could impact early neural differentiation that confirmed by the RNA-Seq results of neural progenitor cells (NPCs). The increased apoptosis and accelerated NPC differentiation in KI iPSCs are associated with excessive unfolded protein response (UPR) pathway activation,and can be rescued by UPR pathway inhibitor. Our study reveals potential pathogenic mechanism of GPIBD22 and providing new insights into the therapeutic strategy for GPIBD. View Publication

ABSTRACTHuman induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However,their differentiation into specific cell lineages,such as definitive endoderm (DE) and pancreatic progenitor (PP),often suffers from poor reproducibility,due partially to their pluripotency. In this work,we investigated the impact of iPSC confluency during cell self?renewal and seeding density on cell metabolic activity,glycolysis to oxidative phosphorylation shift,and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual?positive cells in PPs. Interestingly,we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. Graphical Abstract and Lay SummaryHuman induced pluripotent stem cell (iPSC) differentiation into specific cell lineages often shows poor reproducibility due to cell pluripotency. This study demonstrated impact of iPSC seeding strategy on metabolic activity and differentiation potential,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. View Publication

SummaryGlioblastomas are the most common malignant brain tumors in adults; they are highly aggressive and heterogeneous and show a high degree of plasticity. Here,we show that methyltransferase-like 7B (METTL7B) is an essential regulator of lineage specification in glioblastoma,with an impact on both tumor size and invasiveness. Single-cell transcriptomic analysis of these tumors and of cerebral organoids derived from expanded potential stem cells overexpressing METTL7B reveal a regulatory role for the gene in the neural stem cell-to-astrocyte differentiation trajectory. Mechanistically,METTL7B downregulates the expression of key neuronal differentiation players,including SALL2,via post-translational modifications of histone marks. Graphical abstract Highlights•METTL7B is highly expressed in human glioblastoma stem cells•METTL7B regulates tumor size and invasiveness in an in vivo xenograft model•METTL7B controls the neural stem cell-to-astrocyte differentiation trajectory•METTL7B regulates SALL2 expression via H3K27me3 modulation Constantinou et al. identify METTL7B as an essential regulator of lineage specification and a modulator of the expression of the transcription factor SALL2 with wide-ranging impacts on invasion and tumor growth in glioblastoma. View Publication

Stargardt disease (STGD),predominantly caused by mutations in the ABCA4 gene,is a leading cause of inherited retinal degeneration. Although several lines of mice expressing disease-causing variants have been produced,mice due to the lack of macular may not be the perfect model to mimic the characteristics of STGD. To address this knowledge gap,we generated retinal organoids from patient-derived induced pluripotent stem cells (iPSCs) harboring ABCA4 mutations and performed biological validation. The generated retinal organoids were subjected to single-cell RNA sequencing (scRNA-seq) at major developmental stages (40,90,150,200,and 260 days),and we additionally compared the transcriptomics with our recently published control retinal organoids to further confirm the reliability of the dataset. By using iPSCs carrying most common variant in Chinese STGD patients,the dataset not only provides a powerful resource for studying STGD,but also offers novels insight into the developmental mechanisms underlying ABCA4-associated pathological changes in the retinal organoid system. View Publication

Disease modeling with isogenic Induced Pluripotent Stem Cell (iPSC)-differentiated organoids serves as a powerful technique for studying disease mechanisms. Multiplexed coculture is crucial to mitigate batch effects when studying the genetic effects of disease-causing variants in differentiated iPSCs or organoids,and demultiplexing at the single-cell level can be conveniently achieved by assessing natural genetic barcodes. Here,to enable cost-efficient time-series experimental designs via multiplexed bulk and single-cell RNA-seq of hybrids,we introduce a computational method in our Vireo Suite,Vireo-bulk,to effectively deconvolve pooled bulk RNA-seq data by genotype reference,and thereby quantify donor abundance over the course of differentiation and identify differentially expressed genes among donors. Furthermore,with multiplexed scRNA-seq and bulk RNA-seq,we demonstrate the usefulness and necessity of a pooled design to reveal donor iPSC line heterogeneity during macrophage cell differentiation and to model rare WT1 mutation-driven kidney disease with chimeric organoids. Our work provides an experimental and analytic pipeline for dissecting disease mechanisms with chimeric organoids. IPSC-derived organoids model diseases. Multiplexed coculture and demultiplexing natural genetic barcodes aid in studying genetic effects. Here,authors introduce Vireo-bulk to deconvolve bulk RNA-seq data,quantify donor abundance and identify differentially expressed genes. View Publication

Human genetics analysis has identified many noncoding SNPs associated with diabetic traits,but whether and how these variants contribute to diabetes is largely unknown. Here,we focus on a noncoding variant,rs6048205,and report that the risk-G variant impairs the generation of PDX1+/NKX6-1+ pancreatic progenitor cells and further results in the abnormal decrease of functional ? cells during pancreatic differentiation. Mechanistically,this risk-G variant greatly enhances RXRA binding and over-activates FOXA2 transcription,specifically in the pancreatic progenitor stage,which in turn represses NKX6-1 expression. Consistently,inducible FOXA2 overexpression could phenocopy the differentiation defect. More importantly,mice carrying risk-G exhibit abnormal pancreatic islet architecture and are more sensitive to streptozotocin or a high-fat diet to develop into diabetes eventually. This study not only identifies a causal noncoding variant in diabetes susceptibility but also dissects the underlying gain-of-function mechanism by recruiting stage-specific factors. How GWAS-annotated noncoding SNPs contribute to diabetes remains unclear. Here,the authors report that the noncoding SNP rs6048205 drives stage-specific defects in human pancreatic differentiation and increases diabetes susceptibility in mice. View Publication

The emergence of drug-resistant Mycobacterium tuberculosis (M.tb) has led to the development of novel anti-tuberculosis (anti-TB) drugs. Common methods for testing the efficacy of new drugs,including two-dimensional cell culture models or animal models,have several limitations. Therefore,an appropriate model representative of the human organism is required. Here,we developed an M.tb infection model using human lung organoids (hLOs) and demonstrated that M.tb H37Rv can infect lung epithelial cells and human macrophages (hM?s) in hLOs. This novel M.tb infection model can be cultured long-term and split several times while maintaining a similar number of M.tb H37Rv inside the hLOs. Anti-TB drugs reduced the intracellular survival of M.tb in hLOs. Notably,M.tb growth in hLOs was effectively suppressed at each passage by rifampicin and bedaquiline. Furthermore,a reduction in inflammatory cytokine production and intracellular survival of M.tb were observed upon knockdown of MFN2 and HERPUD1 (host-directed therapeutic targets for TB) in our M.tb H37Rv-infected hLO model. Thus,the incorporation of hM?s and M.tb into hLOs provides a powerful strategy for generating an M.tb infection model. This model can effectively reflect host-pathogen interactions and be utilized to test the efficacy of anti-TB drugs and host-directed therapies. Author summaryEstablishment of M.tb infection model is imperative to develop new anti-TB drugs based on the pathogenesis of TB. Various animal models,including mice,rats,guinea pigs,non-human primates,rabbits,cattle,and zebrafish,are commonly used in TB research to mimic TB symptoms and study immune responses to M.tb infection. In vitro models,such as agent-based models allow examination of host-pathogen interactions,early granuloma formation and drug screening,providing cellular-level insights. However,these models may not fully represent human immunopathology owing to differences in immune cell distributions. Lung organoids mimic human lung dynamics and functions,providing crucial insights into immune responses to TB. In this study,an M.tb infection model developed using hLOs demonstrated infection of lung epithelial cells and human macrophages,reflecting host-pathogen interactions. This model is attractive for evaluating the efficacy of anti-TB drugs and host-directed therapies. View Publication

SummaryThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS),a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits,ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes,little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/? induced pluripotent stem cells to model CNCC specification,we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT),a process necessary for CNCC delamination and migration from the neural tube. Furthermore,wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level,these EMT enhancers contain binding motifs for ZIC2,and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers,ZIC2 relocates to neuronal enhancers,triggering aberrant neuronal gene activation. In mice,deletion of Zic2 impairs NCC delamination,while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. Graphical abstract ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition,a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers,resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. View Publication

To uncover molecular changes underlying blood-brain-barrier dysfunction in Alzheimer’s disease,we performed single nucleus RNA sequencing in 24 Alzheimer’s disease and control brains and focused on vascular and astrocyte clusters as main cell types of blood-brain-barrier gliovascular-unit. The majority of the vascular transcriptional changes were in pericytes. Of the vascular molecular targets predicted to interact with astrocytic ligands,SMAD3,upregulated in Alzheimer’s disease pericytes,has the highest number of ligands including VEGFA,downregulated in Alzheimer’s disease astrocytes. We validated these findings with external datasets comprising 4,730 pericyte and 150,664 astrocyte nuclei. Blood SMAD3 levels are associated with Alzheimer’s disease-related neuroimaging outcomes. We determined inverse relationships between pericytic SMAD3 and astrocytic VEGFA in human iPSC and zebrafish models. Here,we detect vast transcriptome changes in Alzheimer’s disease at the gliovascular-unit,prioritize perturbed pericytic SMAD3-astrocytic VEGFA interactions,and validate these in cross-species models to provide a molecular mechanism of blood-brain-barrier disintegrity in Alzheimer’s disease. Systematic studies are needed to discover molecular determinants of blood brain barrier dysfunction in Alzheimer’s disease. This study identifies perturbed pericytic SMAD3-astrocytic VEGFA interactions as a potential driver of this dysfunction. View Publication

Diabetes involves the death or dysfunction of pancreatic ?-cells. Analysis of bulk sequencing from human samples and studies using in vitro and in vivo models suggest that endoplasmic reticulum and inflammatory signaling play an important role in diabetes progression. To better characterize cell type-specific stress response,we perform multiplexed single-cell RNA sequencing to define the transcriptional signature of primary human islet cells exposed to endoplasmic reticulum and inflammatory stress. Through comprehensive pair-wise analysis of stress responses across pancreatic endocrine and exocrine cell types,we define changes in gene expression for each cell type under different diabetes-associated stressors. We find that ?-,?-,and ductal cells have the greatest transcriptional response. We utilize stem cell-derived islets to study islet health through the candidate gene CIB1,which was upregulated under stress in primary human islets. Our findings provide insights into cell type-specific responses to diabetes-associated stress and establish a resource to identify targets for diabetes therapeutics. Endoplasmic reticulum and inflammatory stress are associated with diabetes. Maestas et al. use single-cell sequencing to profile primary human islets under stress and identified tissue and cell-type responses. View Publication

SummaryPattern recognition receptors (PRRs) induce host defense but can also induce exacerbated inflammatory responses. This raises the question of whether other mechanisms are also involved in early host defense. Using transcriptome analysis of disrupted transcripts in herpes simplex virus (HSV)-infected cells,we find that HSV infection disrupts the hypoxia-inducible factor (HIF) transcription network in neurons and epithelial cells. Importantly,HIF activation leads to control of HSV replication. Mechanistically,HIF activation induces autophagy,which is essential for antiviral activity. HSV-2 infection in vivo leads to hypoxia in CNS neurons,and mice with neuron-specific HIF1/2? deficiency exhibit elevated viral load and augmented PRR signaling and inflammatory gene expression in the CNS after HSV-2 infection. Data from human stem cell-derived neuron and microglia cultures show that HIF also exerts antiviral and inflammation-restricting activity in human CNS cells. Collectively,the HIF transcription factor system senses virus-induced hypoxic stress to induce cell-intrinsic antiviral responses and limit inflammation. Graphical abstract Highlights•HSV-1 and -2 disrupt the hypoxia-inducible factor (HIF) network in permissive cells•HIF activation induces autophagy,which exerts anti-HSV activity in neurons•Neuronal HIF activation regulates infection and inflammation in the infected brain Using transcriptome analysis of disrupted transcripts in herpes simplex virus-infected cells,Farahani et al. identify the hypoxia-inducible factor gene network to possess antiviral activity through induction of autophagy. This contributes to antiviral defense and regulation of inflammation during infection in the CNS. View Publication