技术资料

Neurological damage caused by peripheral nerve injury can be devastating and is a common neurological disorder that,along with muscle disorders,reduces the quality of life. Neural crest cells (NCCs) are a transient cell population that occurs during embryogenesis,can differentiate into various cells upon transplantation,and has potential therapeutic effects on neurological diseases. However,there are limitations to cell therapy,such as uncontrolled differentiation and tumor formation. Extracellular vesicles (EVs),which are non-cellular potential therapeutic candidates,are nanosized membrane-bound vesicles. Studies have been reported using starch cells derived from neural cells,such as neural stem cells,because they are involved in cell-to-cell communication and carry a variety of bioactive molecules. We investigated the effects of EVs isolated from NCCs on neuronal cell death and inflammation. Additionally,we overexpressed the nerve growth factor (NGF),which is involved in neural cell growth and proliferation,in NCCs to further investigate the effects of EVs containing NGF. NCCoe-NGF-EVs showed neuroprotective and regenerative properties by modulating inflammatory pathway,promoting Schwann cell activation,and enhancing remyelination. In vitro studies on NCCoe-NGF-EVs suppressed pro-inflammatory cytokines and reduced oxidative stress-induced neuronal apoptosis through NF-?B pathway inhibition and ERK,AKT signal activation. We also evaluated the effect of EVs on neuropathy by performing in vivo study. Our results suggest that NCCoe-NGF-EV had neuroprotective effects by reducing neuronal apoptosis and promoting neuronal proliferation based on neurite outgrowth and anti-inflammation effects treated with NCCoe-NGF-EVs. In addition,NCCoe-NGF-EVs were protected muscle loss caused by peripheral nerve injury. NCCoe-NGF-EV induced regeneration of damaged nerves and inhibited cell death through anti-inflammatory effects. This study suggests the potential of NGF-enriched EVs as non-cellular therapeutic platform for peripheral neuropathies and other neuroinflammatory disorders. 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

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

Organoids,derived from human induced pluripotent stem cells (hiPSCs),are intricate three-dimensional in vitro structures that mimic many key aspects of the complex morphology and functions of in vivo organs such as the retina and heart. Traditional histological methods,while crucial,often fall short in analyzing these dynamic structures due to their inherently static and destructive nature. In this study,we leveraged the capabilities of optical coherence tomography (OCT) for rapid,non-invasive imaging of both retinal,cerebral,and cardiac organoids. Complementing this,we developed a sophisticated deep learning approach to automatically segment the organoid tissues and their internal structures,such as hollows and chambers. Utilizing this advanced imaging and analysis platform,we quantitatively assessed critical parameters,including size,area,volume,and cardiac beating,offering a comprehensive live characterization and classification of the organoids. These findings provide profound insights into the differentiation and developmental processes of organoids,positioning quantitative OCT imaging as a potentially transformative tool for future organoid research. View Publication

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants,as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast,larger SVs,which may significantly influence human phenotypes,have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study,we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines,which were not identified by long-read sequencing. Additionally,various exon deletions were confirmed by OGM in the PRKN gene of iPSCs,of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs,no gene fusions,no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary,OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques,which is supportive for OGM’s future use in gene discovery and iPSC line screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-024-10902-1. View Publication

The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications,as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines,robustly identifies inefficient gRNAs across different published datasets,and is easily accessible via online genome browser tracks. In addition,we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a,which may be relevant for safety in gene therapy applications. Reliable prediction of guide RNA (gRNA) activity is key for efficient CRISPR gene editing. Here,the authors show that efficiency of gRNAs is often underestimated when only indels are scored and introduce tools for predicting activity of chemically synthesized gRNAs and HDR efficiency. View Publication

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor screens have enabled efficient production of a few cell types; however,engineering cell types that require complex transcription factor combinations remains challenging. Here,we report an iterative,high-throughput single-cell transcription factor screening method that enables the identification of transcription factor combinations for specialized cell differentiation,which we validated by differentiating human microglia-like cells. We found that the expression of six transcription factors,SPI1,CEBPA,FLI1,MEF2C,CEBPB,and IRF8,is sufficient to differentiate human iPSC into cells with transcriptional and functional similarity to primary human microglia within 4 days. Through this screening method,we also describe a novel computational method allowing the exploration of single-cell RNA sequencing data derived from transcription factor perturbation assays to construct causal gene regulatory networks for future cell fate engineering. Liu et al. developed a platform to identify transcription factors (TFs) that turn stem cells into desired cell types. They discovered six key TFs that produce microglia efficiently,enhancing cell differentiation methods. View Publication

Brain organoids offer unprecedented insights into brain development and disease modeling and hold promise for drug screening. Significant hindrances,however,are morphological and cellular heterogeneity,inter-organoid size differences,cellular stress,and poor reproducibility. Here,we describe a method that reproducibly generates thousands of organoids across multiple hiPSC lines. These High Quantity brain organoids (Hi-Q brain organoids) exhibit reproducible cytoarchitecture,cell diversity,and functionality,are free from ectopically active cellular stress pathways,and allow cryopreservation and re-culturing. Patient-derived Hi-Q brain organoids recapitulate distinct forms of developmental defects: primary microcephaly due to a mutation in CDK5RAP2 and progeria-associated defects of Cockayne syndrome. Hi-Q brain organoids displayed a reproducible invasion pattern for a given patient-derived glioma cell line. This enabled a medium-throughput drug screen to identify Selumetinib and Fulvestrant,as inhibitors of glioma invasion in vivo. Thus,the Hi-Q approach can easily be adapted to reliably harness brain organoids’ application for personalized neurogenetic disease modeling and drug discovery. Human brain organoids are plagued by heterogeneity and poor reproducibility,critical parameters for reliable disease modeling and drug testing. Here,the authors report on Hi-Q organoids which solve these limitations and can be cryopreserved in large quantities. View Publication

SUMMARY Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis,how ALK contributes to tumor formation remains unclear. Here,we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic,while ALK/MYCN tumors resembled mesenchymal,neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling,particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN,but not FN1,delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore,loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally,inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus,ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling. In brief Huang et al. used a human stem cell model to elucidate the mechanism for cooperation between MYCN and ALK. ALK contributes to tumor growth by upregulating the extracellular matrix protein periostin and activating WNT signaling. Periostin and WNT signal through a feedforward loop. Graphical Abstract View Publication

Doublecortin is a neuronal microtubule-associated protein that regulates microtubule structure in neurons. Mutations in Doublecortin cause lissencephaly and subcortical band heterotopia by impairing neuronal migration. We use CRISPR/Cas9 to knock-out the Doublecortin gene in induced pluripotent stem cells and differentiate the cells into cortical neurons. DCX-KO neurons show reduced velocities of nuclear movements and an increased number of neurites early in neuronal development,consistent with previous findings. Neurite branching is regulated by a host of microtubule-associated proteins,as well as by microtubule polymerization dynamics. However,EB comet dynamics are unchanged in DCX-KO neurons. Rather,we observe a significant reduction in ?-tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching are rescued by expression of Doublecortin or of TTLL11,an ?-tubulin glutamylase. Using U2OS cells as an orthogonal model system,we show that DCX and TTLL11 act synergistically to promote polyglutamylation. We propose that Doublecortin acts as a positive regulator of ?-tubulin polyglutamylation and restricts neurite branching. Our results indicate an unexpected role for Doublecortin in the homeostasis of the tubulin code. Lissencephaly is a severe neurodevelopmental disease often caused by mutations in the Dcx gene. Using a human cellular model of lissencephaly,the authors report that DCX restricts neuronal branching by activating tubulin polyglutamylation. View Publication

SUMMARY Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19,HIV,and apolipoprotein-L1 (APOL1) nephropathy,but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids,primary endothelial cells,and patient samples,we demonstrate that IFN-? induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing,immunoblotting,and quantitative fluorescence-based assays reveal that IFN-?-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-? signaling inhibits APOL1 expression,prevents upregulation of pyroptosis-associated genes,and rescues vascular networks. Multiomic analyses in patients with COVID-19,proteinuric kidney disease,and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-? signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis,suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. In brief Juliar et al. address interferon signaling in kidney disease. Organoids,primary cells,and clinical datasets reveal that interferon signaling simultaneously induces APOL1 expression and endothelial cell pyroptosis. This suggests a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. The findings may also be relevant in other organs. Graphical Abstract View Publication

SummaryDysfunction of the sympathetic nervous system and increased epicardial adipose tissue (EAT) have been independently associated with the occurrence of cardiac arrhythmia. However,their exact roles in triggering arrhythmia remain elusive. Here,using an in vitro coculture system with sympathetic neurons,cardiomyocytes,and adipocytes,we show that adipocyte-derived leptin activates sympathetic neurons and increases the release of neuropeptide Y (NPY),which in turn triggers arrhythmia in cardiomyocytes by interacting with the Y1 receptor (Y1R) and subsequently enhancing the activity of the Na+/Ca2+ exchanger (NCX) and calcium/calmodulin-dependent protein kinase II (CaMKII). The arrhythmic phenotype can be partially blocked by a leptin neutralizing antibody or an inhibitor of Y1R,NCX,or CaMKII. Moreover,increased EAT thickness and leptin/NPY blood levels are detected in atrial fibrillation patients compared with the control group. Our study provides robust evidence that the adipose-neural axis contributes to arrhythmogenesis and represents a potential target for treating arrhythmia. Graphical abstract Highlights•Stem cell-based coculture model can simulate the pathogenesis of cardiac arrhythmia•The adipose-neural axis plays critical roles in cardiac arrhythmias•Leptin,NPY/Y1R,NCX,and CaMKII are potential intervention targets for arrhythmia•Increased EAT thickness and leptin/NPY levels are detected in CS blood of AF patients Fan et al. establish a stem cell-based coculture model to mimic the in vivo cardiac microenvironment and elucidate that the adipose-neural interaction plays a critical role in epicardial adipose tissue-related cardiac arrhythmia through leptin-NPY axis. Their results may provide potential therapeutic targets for treating arrhythmia. View Publication