参考文献

PIEZO1 is critical to numerous physiological processes,transducing diverse mechanical stimuli into electrical and chemical signals. Recent studies underscore the importance of visualizing endogenous PIEZO1 activity and localization to understand its functional roles. To enable physiologically and clinically relevant studies on human PIEZO1,we genetically engineered human induced pluripotent stem cells (hiPSCs) to express a HaloTag fused to endogenous PIEZO1. Combined with advanced imaging,our chemogenetic platform allows precise visualization of PIEZO1 localization dynamics in various cell types. Furthermore,the PIEZO1-HaloTag hiPSC technology facilitates the non-invasive monitoring of channel activity across diverse cell types using Ca2+-sensitive HaloTag ligands,achieving temporal resolution approaching that of patch clamp electrophysiology. Finally,we use lightsheet microscopy on hiPSC-derived neural organoids to achieve molecular scale imaging of PIEZO1 in three-dimensional tissue. Our advances establish a platform for studying PIEZO1 mechanotransduction in human systems,with potential for elucidating disease mechanisms and targeted drug screening. PIEZO1 is critical in numerous physiological processes,but monitoring its activity and localization in cells can be challenging. Here,the authors present a chemogenetic platform to visualize endogenous human PIEZO1 localization and activity in native cellular conditions,expanding the knowledge on mechanotransduction across single cells and tissue organoids. View Publication

PurposeDilated cardiomyopathy (DCM) is a prevalent form of heart failure with limited therapeutic options. This study explores a novel treatment strategy involving the delivery of exosome-derived miRNA-185-5p inhibitors encapsulated in liposomes,aiming to target cardiac tissue and alleviate myocardial apoptosis and cuproptosis in DCM.MethodsThe miRNA-185-5p inhibitor,identified in our previous study and extracted from exosomes,was encapsulated in liposomes functionalized with a cardiac-targeting peptide. This system was used in both in vitro and in vivo models of DCM induced by doxorubicin (DOX). We evaluated the effects of this treatment on cardiac function,apoptosis,cuproptosis,oxidative stress,and fibrosis using echocardiography,histological analysis,Western blotting,and biochemical assays.ResultsIn vitro experiments demonstrated that the Lipo@miR-185-5p inhibitor markedly attenuated apoptosis and cuproptosis in H9C2 cells and iPSC-derived cardiomyocytes,with a 42.6% reduction in apoptotic cell rates and over 50% downregulation of cuproptosis-related markers (both P < 0.01). In vivo,the targeted liposomal formulation significantly improved cardiac function in DOX-induced DCM mice,as evidenced by a 27.3% increase in left ventricular ejection fraction (LVEF) and a 36.5% reduction in myocardial fibrosis area (P < 0.01),along with enhanced survival. These findings underscore the therapeutic potential of this targeted delivery strategy for the treatment of dilated cardiomyopathy.ConclusionLipo@miR-185-5p inhibitor,utilizing exosome-derived miRNA and targeted liposomal delivery,effectively alleviates DCM-induced myocardial dysfunction. This approach represents a promising therapeutic strategy for cardiovascular diseases by targeting specific molecular mechanisms involved in heart failure. View Publication

SummaryApolipoprotein E4 (APOE4) is the leading genetic risk factor for Alzheimer’s disease. While most studies examine the role of APOE4 in aging,APOE4 causes persistent changes in brain structure as early as infancy and is associated with altered functional connectivity that extends beyond adolescence. Here,we used human induced pluripotent stem cell-derived cortical and ganglionic eminence organoids (COs and GEOs) to examine APOE4’s influence during the development of cortical excitatory and inhibitory neurons. We show that APOE4 reduces cortical neurons and increases glia by promoting gliogenic transcriptional programs. In contrast,APOE4 increases proliferation and differentiation of GABAergic progenitors resulting in early and persistent increases in GABAergic neurons. Multi-electrode array recordings in assembloids revealed that APOE4 disrupts neural network function resulting in heightened excitability and synchronicity. Together,our data provide new insights on how APOE4 influences cortical neurodevelopmental processes and the establishment of functional networks. Highlights•APOE4 accelerates differentiation and maturation at developmental time points•APOE4 results in a loss of cortical neurons and increase in astrocytes and outer radial glia•APOE4 enhances proliferation,differentiation,and maturation of GABAergic neurons•APOE4 alters GABA-related genes,linked to increased GABA response and synchronicity Meyer-Acosta et al. reveal that Alzheimer’s disease genetic risk factor APOE4 decreases cortical neurons and increases glia in cortical organoids and enhances GABAergic neuron maturation in ganglionic eminence organoids derived from iPSCs. These cellular changes result in heightened excitability and synchronicity in APOE4-fused organoids,providing insight into the cellular processes that may underlie altered brain structure observed in APOE4 infants. View Publication

Alternative splicing is a major contributor of transcriptomic complexity,but the extent to which transcript isoforms are translated into stable,functional protein isoforms is unclear. Furthermore,detection of relatively scarce isoform-specific peptides is challenging,with many protein isoforms remaining uncharted due to technical limitations. Recently,a family of advanced targeted MS strategies,termed internal standard parallel reaction monitoring (IS-PRM),have demonstrated multiplexed,sensitive detection of pre-defined peptides of interest. Such approaches have not yet been used to confirm existence of novel peptides. Here,we present a targeted proteogenomic approach that leverages sample-matched long-read RNA sequencing (LR RNAseq) data to predict potential protein isoforms with prior transcript evidence. Predicted tryptic isoform-specific peptides,which are specific to individual gene product isoforms,serve as “triggers” and “targets” in the IS-PRM method,Tomahto. Using the model human stem cell line WTC11,LR RNAseq data were generated and used to inform the generation of synthetic standards for 192 isoform-specific peptides (114 isoforms from 55 genes). These synthetic “trigger” peptides were labeled with super heavy tandem mass tags (TMT) and spiked into TMT-labeled WTC11 tryptic digest,predicted to contain corresponding endogenous “target” peptides. Compared to DDA mode,Tomahto increased detectability of isoforms by 3.6-fold,resulting in the identification of five previously unannotated isoforms. Our method detected protein isoform expression for 43 out of 55 genes corresponding to 54 resolved isoforms. This LR RNA seq-informed Tomahto targeted approach,called LRP-IS-PRM,is a new modality for generating protein-level evidence of alternative isoforms – a critical first step in designing functional studies and eventually clinical assays. View Publication

Background: Effective molecular diagnosis of congenital diseases hinges on comprehensive genomic analysis,traditionally reliant on various methodologies specific to each variant type-whole exome or genome sequencing for single nucleotide variants (SNVs),array CGH for copy-number variants (CNVs),and microscopy for structural variants (SVs). Methods: We introduce a novel,integrative approach combining exome sequencing with chromosome conformation capture,termed Exo-C. This method enables the concurrent identification of SNVs in clinically relevant genes and SVs across the genome and allows analysis of heterozygous and mosaic carriers. Enhanced with targeted long-read sequencing,Exo-C evolves into a cost-efficient solution capable of resolving complex SVs at base-pair accuracy. Results: Applied to 66 human samples Exo-C achieved 100% recall and 73% precision in detecting chromosomal translocations and SNVs. We further benchmarked its performance for inversions and CNVs and demonstrated its utility in detecting mosaic SVs and resolving diagnostically challenging cases. Conclusions: Through several case studies,we demonstrate how Exo-C's multifaceted application can effectively uncover diverse causative variants and elucidate disease mechanisms in patients with rare disorders. View Publication

Peptide-based supramolecular nanostructures offer a versatile platform with substantial promise for clinical translation in regenerative medicine. These systems allow for the incorporation of biologically active sequences and can be engineered to modulate tissue-specific parameters such as stiffness,diffusivity,and biodegradability. We developed here a bioactive supramolecular nanostructure containing a peptide designed based on glial cell-derived neurotrophic factor. These nanostructures form scaffolds that mimic important trophic effects provided by this growth factor on iPSC-derived human dopaminergic neurons. Our in vitro data show that the nanostructures promote cell viability,confer neuroprotection against 6-hydroxydopamine toxicity,enhance neuronal morphology,facilitate electrophysiological maturation,and induce genes involved in neuronal survival. We also found that the scaffold promoted axonal extension in midbrain human organoids. These findings suggest that the supramolecular system could be useful to improve outcomes in cell-based therapies for Parkinson’s disease,where progressive dopaminergic degeneration is a hallmark. View Publication

SummaryThe ch12q13 locus is among the most significant childhood obesity loci identified in genome-wide association studies. This locus resides in a non-coding region within FAIM2; thus,the underlying causal variant(s) presumably influence disease susceptibility via cis-regulation. We implicated rs7132908 as a putative causal variant by leveraging our in-house 3D genomic data and public domain datasets. Using a luciferase reporter assay,we observed allele-specific cis-regulatory activity of the immediate region harboring rs7132908. We generated isogenic human embryonic stem cell lines homozygous for either rs7132908 allele to assess changes in gene expression and chromatin accessibility throughout a differentiation to hypothalamic neurons,a key cell type known to regulate feeding behavior. The rs7132908 obesity risk allele influenced expression of FAIM2 and other genes and decreased the proportion of neurons produced by differentiation. We have functionally validated rs7132908 as a causal obesity variant that temporally regulates nearby effector genes and influences neurodevelopment and survival. Graphical abstract Highlights•rs7132908 is a causal variant at the chr12q13 obesity locus•rs7132908 regulates nearby effector genes with allele and cell-type specificity•Obesity risk allele decreases generation of neurons that regulate appetite A locus on chr12q13 is strongly associated with childhood obesity by genome-wide associate studies. Littleton et al. identified a causal variant at this locus,which regulates gene expression in neural cell types. The obesity risk allele also decreased the proportion of appetite-regulating hypothalamic neurons generated by stem cell differentiation. View Publication

For genome editing,the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins,which enable the targeted manipulation of transcriptomes and epigenomes,remain significantly less accessible. Here,we describe the production,delivery,and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency,even for genes that are strongly silenced,such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies,gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing,enabling undiminished gene activation of multiple genes simultaneously. Applying these insights,we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally,we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors. View Publication

Human development relies on the correct replication,maintenance and segregation of our genetic blueprints. How these processes are monitored across embryonic lineages,and why genomic mosaicism varies during development remain unknown. Using pluripotent stem cells,we identify that several patterning signals—including WNT,BMP,and FGF—converge into the modulation of DNA replication stress and damage during S-phase,which in turn controls chromosome segregation fidelity in mitosis. We show that the WNT and BMP signals protect from excessive origin firing,DNA damage and chromosome missegregation derived from stalled forks in pluripotency. Cell signalling control of chromosome segregation declines during lineage specification into the three germ layers,but re-emerges in neural progenitors. In particular,we find that the neurogenic factor FGF2 induces DNA replication stress-mediated chromosome missegregation during the onset of neurogenesis,which could provide a rationale for the elevated chromosomal mosaicism of the developing brain. Our results highlight roles for morphogens and cellular identity in genome maintenance that contribute to somatic mosaicism during mammalian development. Here the authors show that the patterning signals WNT,BMP,and FGF control chromosome segregation fidelity during early lineage specification and neurogenesis,which could provide a rationale for the spatio-temporal distribution of genomic mosaicism during human development. View Publication

SummaryCardiovascular disease (CVD) is associated with both genetic variants and environmental factors. One unifying consequence of the molecular risk factors in CVD is DNA damage,which must be repaired by DNA damage response proteins. However,the impact of DNA damage on global cardiomyocyte protein abundance,and its relationship to CVD risk remains unclear. We therefore treated induced pluripotent stem cell-derived cardiomyocytes with the DNA-damaging agent Doxorubicin (DOX) and a vehicle control,and identified 4,178 proteins that contribute to a network comprising 12 co-expressed modules and 403 hub proteins with high intramodular connectivity. Five modules correlate with DOX and represent distinct biological processes including RNA processing,chromatin regulation and metabolism. DOX-correlated hub proteins are depleted for proteins that vary in expression across individuals due to genetic variation but are enriched for proteins encoded by loss-of-function intolerant genes. While proteins associated with genetic risk for CVD,such as arrhythmia are enriched in specific DOX-correlated modules,DOX-correlated hub proteins are not enriched for known CVD risk proteins. Instead,they are enriched among proteins that physically interact with CVD risk proteins. Our data demonstrate that DNA damage in cardiomyocytes induces diverse effects on biological processes through protein co-expression modules that are relevant for CVD,and that the level of protein connectivity in DNA damage-associated modules influences the tolerance to genetic variation. View Publication