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BackgroundRNA-binding proteins (RBPs) are essential in cardiac development. However,a large of them have not been characterized during the process.MethodsWe applied the human embryonic stem cells (hESCs) differentiated into cardiomyocytes model and constructed SAMD4A-knockdown/overexpression hESCs to investigate the role of SAMD4A in cardiomyocyte lineage specification.ResultsSAMD4A,an RBP,exhibits increased expression during early heart development. Suppression of SAMD4A inhibits the proliferation of hESCs,impedes cardiac mesoderm differentiation,and impairs the function of hESC-derived cardiomyocytes. Correspondingly,forced expression of SAMD4A enhances proliferation and promotes cardiomyogenesis. Mechanistically,SAMD4A specifically binds to FGF2 via a specific CNGG/CNGGN motif,stabilizing its mRNA and enhancing translation,thereby upregulating FGF2 expression,which subsequently modulates the AKT signaling pathway and regulates cardiomyocyte lineage differentiation. Additionally,supplementation of FGF2 can rescue the proliferation defect of hESCs in the absence of SAMD4A.ConclusionsOur study demonstrates that SAMD4A orchestrates cardiomyocyte lineage commitment through the post-transcriptional regulation of FGF2 and modulation of AKT signaling. These findings not only underscore the essential role of SAMD4A in cardiac organogenesis,but also provide critical insights into the molecular mechanisms underlying heart development,thereby informing potential therapeutic strategies for congenital heart disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04269-7. View Publication

Organoids are self-assembled 3D cellular structures that resemble organs structurally and functionally,providing in vitro platforms for molecular and therapeutic studies. Generation of organoids from human cells often requires long and costly procedures with arguably low efficiency. Prediction and selection of cellular aggregates that result in healthy and functional organoids can be achieved by using artificial intelligence-based tools. Transforming images of 3D cellular constructs into digitally processable data sets for training deep learning models requires labeling of morphological boundaries,which often is performed manually. Here,we report an application named OrganoLabeler,which can create large image-based data sets in a consistent,reliable,fast,and user-friendly manner. OrganoLabeler can create segmented versions of images with combinations of contrast adjusting,K-means clustering,CLAHE,binary,and Otsu thresholding methods. We created embryoid body and brain organoid data sets,of which segmented images were manually created by human researchers and compared with OrganoLabeler. Validation is performed by training U-Net models,which are deep learning models specialized in image segmentation. U-Net models,which are trained with images segmented by OrganoLabeler,achieved similar or better segmentation accuracies than the ones trained with manually labeled reference images. OrganoLabeler can replace manual labeling,providing faster and more accurate results for organoid research free of charge. View Publication

Genomic instability is the main cause of abnormal embryo development and abortion. NLRP7 dysfunctions affect embryonic development and lead to Hydatidiform Moles,but the underlying mechanisms remain largely elusive. Here,we show that NLRP7 knockout affects the genetic stability,resulting in increased DNA damage in both human embryonic stem cells and blastoids,making embryonic cells in blastoids more susceptible to apoptosis. Mechanistically,NLRP7 can interact with factors related to alternative splicing and DNA damage response,including DDX39B,PRPF8,THRAP3 and PARP1. Moreover,NLRP7 dysfunction leads to abnormal alternative splicing of genes involved in homologous recombination in human embryonic stem cells,Such as Brca1 and Rad51. These results indicate that NLRP7-mediated Alternative splicing is potentially required for the maintenance of genome integrity during early human embryogenesis. Together,this study uncovers that NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes. NLRP7 plays an essential role in the maintenance of genetic stability during early human embryonic development by regulating alternative splicing of homologous recombination-related genes. View Publication

BackgroundPathological deposition of hyperphosphorylated tau in the brain closely correlates with the course of Alzheimer’s disease (AD). Tau pathology occurs in axons of affected neurons and tau removal from axons might thus be an early intervention strategy.MethodsWe investigated the role of the RNA-binding protein hnRNP R in axonal localization and local translation of Mapt mRNA in neurons cultured from hnRNP R knockout mice. hnRNP R knockout mice were crossed with 5×FAD mice,an AD mouse model,and the effects of hnRNP R loss on the deposition of phospho-tau and amyloid-? plaques were evaluated. We designed antisense oligonucleotides (MAPT-ASOs) to block the binding of hnRNP R to Mapt mRNA. Cultured mouse and human neurons were treated with MAPT-ASOs and axonal Mapt mRNA and tau protein levels were quantified. MAPT-ASO was injected intracerebroventricularly into 5×FAD mice followed by quantification of phospho-tau aggregates and amyloid-? plaques in their brains. Protein changes in brains of 5×FAD mice treated with the MAPT-ASO were measured by mass spectrometry.ResultsMapt mRNA and tau protein were reduced in axons but not cell bodies of primary neurons cultured from hnRNP R knockout mice. Brains of 5×FAD mice deficient for hnRNP R contained less phospho-tau aggregates and amyloid-? plaques in the cortex and hippocampus. Treatment of neurons with MAPT-ASOs to block hnRNP R binding to Mapt similarly reduced axonal tau levels. Intracerebroventricular injection of a MAPT-ASO reduced the phospho-tau and plaque load and prevented neurodegeneration in the brains of 5×FAD mice,accompanied by rescue of proteome alterations.ConclusionLowering of tau selectively in axons thus represents an innovative therapeutic perspective for treatment of AD and other tauopathies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40035-025-00499-0. View Publication

Cell fate progression of pluripotent progenitors is strictly regulated,resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study,we use human brain and retina organoid models and present single-cell profiling of H3K27ac,H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage,providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction,resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination. The mechanisms underlying human cell diversity are unclear. Here the authors provide a single-cell epigenome map of human neural organoid development and dissect how epigenetic changes control cell fate specification from pluripotency to distinct cerebral and retina neural types. View Publication

There has been a recent drive to replace in vivo studies with in vitro studies in the field of toxicity testing. Therefore,instead of conventional animal or planar cell culture models,there is an urgent need for in vitro systems whose conditions can be strictly controlled,including cell–cell interactions and sensitivity to low doses of chemicals. Neural organoids generated from human-induced pluripotent stem cells (iPSCs) are a promising in vitro platform for modeling human brain development. In this study,we developed a new tool based on various iPSCs to study and predict chemical-induced toxicity in humans. The model displayed several neurodevelopmental features and showed good reproducibility,comparable to that of previously published models. The results revealed that basic fibroblast growth factor plays a key role in the formation of the embryoid body,as well as complex neural networks and higher-order structures such as layered stacking. Using organoid models,pesticide toxicities were assessed. Cells treated with low concentrations of rotenone underwent apoptosis to a greater extent than those treated with high concentrations of rotenone. Morphological changes associated with the development of neural progenitor cells were observed after exposure to low doses of chlorpyrifos. These findings suggest that the neuronal organoids developed in this study mimic the developmental processes occurring in the brain and nerves and are a useful tool for evaluating drug efficacy,safety,and toxicity. View Publication

SummaryGamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in adults. Depolarizing GABA responses have been well characterized at neuronal-population average level during typical neurodevelopment and partially in brain disorders. However,no investigation has specifically assessed whether a mosaicism of cells with either depolarizing or hyperpolarizing/inhibitory GABAergic responses exists in animals in health/disease at diverse developmental stages,including adulthood. Here,we showed that such mosaicism is present in wild-type (WT) and down syndrome (DS) neuronal networks,as assessed at increasing scales of complexity (cultures,brain slices,behaving mice). Nevertheless,WT mice presented a much lower percentage of cells with depolarizing GABA than DS mice. Restoring the mosaicism of hyperpolarizing and depolarizing GABA-responding neurons to WT levels rescued anxiety behavior in DS mice. Moreover,we found heterogeneous GABAergic responses in developed control and trisomic human induced-pluripotent-stem-cells-derived neurons. Thus,a heterogeneous subpopulation of GABA-responding cells exists in physiological/pathological conditions in mouse and human neurons,possibly contributing to disease-associated behaviors. Graphical abstract Highlights•Subpopulations of GABAAR-responding neurons exist in mouse and human neuronal networks•DS networks exhibit a larger fraction of neurons with depolarizing GABA responses•Restoring physiological GABA-mediated inhibition rescues anxiety behavior in DS mice•Heterogeneous GABAergic responses coexist in control and DS human iPSC neurons Behavioral neuroscience; Developmental neuroscience; Cellular neuroscience View Publication

Arterioles are small blood vessels located just upstream of capillaries in nearly all tissues. Despite the broad and essential role of arterioles in physiology and disease,current knowledge of the functional genomics of arterioles is largely absent. Here,we report extensive maps of chromatin interactions,single-cell expression,and other molecular features in human arterioles and uncover mechanisms linking human genetic variants to gene expression in vascular cells and the development of hypertension. Compared to large arteries,arterioles exhibited a higher proportion of pericytes which were enriched for blood pressure (BP)-associated genes. BP-associated single nucleotide polymorphisms (SNPs) were enriched in chromatin interaction regions in arterioles. We linked BP-associated noncoding SNP rs1882961 to gene expression through long-range chromatin contacts and revealed remarkable effects of a 4-bp noncoding genomic segment on hypertension in vivo. We anticipate that our data and findings will advance the study of the numerous diseases involving arterioles. Liu et al.,report extensive maps of chromatin interactions,single-cell expression,and other molecular features in human arterioles and uncover mechanisms linking noncoding genetic variants to gene expression and the development of hypertension. View Publication

Aging of the brain vasculature plays a key role in the development of neurovascular and neurodegenerative diseases,thereby contributing to cognitive impairment. Among other factors,DNA damage strongly promotes cellular aging,however,the role of genomic instability in brain endothelial cells (EC) and its potential effect on brain homeostasis is still largely unclear. We here investigated how endothelial aging impacts blood-brain barrier (BBB) function by using excision repair cross complementation group 1 (ERCC1)-deficient human brain ECs and an EC-specific Ercc1 knock out (EC-KO) mouse model. In vitro,ERCC1-deficient brain ECs displayed increased senescence-associated secretory phenotype expression,reduced BBB integrity,and higher sprouting capacities due to an underlying dysregulation of the Dll4-Notch pathway. In line,EC-KO mice showed more P21+ cells,augmented expression of angiogenic markers,and a concomitant increase in the number of brain ECs and pericytes. Moreover,EC-KO mice displayed BBB leakage and enhanced cell adhesion molecule expression accompanied by peripheral immune cell infiltration into the brain. These findings were confined to the white matter,suggesting a regional susceptibility. Collectively,our results underline the role of endothelial aging as a driver of impaired BBB function,endothelial sprouting,and increased immune cell migration into the brain,thereby contributing to impaired brain homeostasis as observed during the aging process. View Publication

BackgroundUnderstanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models,lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate,and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule.MethodsTo establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells,we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways,and construct cell lines carrying an inducible NKX3-1 expressing cassette,together with three-dimensional culture system. Unpaired t test was applied for statistical analyses.ResultsWe first successfully generate the definitive endoderm,hindgut,and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1,but fail to express NKX3-1. Therefore,we construct NKX3-1-inducible cell line by homologous recombination,which is eventually able to yield AR,FOXA1,and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally,combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations.ConclusionsThis study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line,as well as provides a stepwise differentiation protocol to generate human prostate-like organoids,which should facilitate the studies on prostate development and disease pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03886-y. View Publication

SummaryNGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study,we present a standardized approach for generating neurons utilizing clonal,targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150 days. Temporal omics,electrophysiological,and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic,imaging,and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally,we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows,profiling data,and functional characterizations enable the development of reproducible human in vitro models of neurological disorders. Graphical abstract Highlights•Optimized NGN2 protocol generates functional postnatal neurons in 28 days•Extensive profiling data provide benchmarks for neuron maturation•Maturation assays reliably assess neuron maturation in single or mixed cell types•Rapid targeted engineering protocol integrates NGN2 into iPSC lines in 3 weeks MotivationUsing induced pluripotent stem cell (iPSC)-derived neurons (iNs) to model diseases requires defined,robust,and reproducible protocols capable of generating predictable neuronal types. In addition,extensive profiling is essential to assess whether iNs are suitable to model specific diseases with desired molecular,functional,and maturation-related features. We sought to establish a standardized protocol for generating iNs at large scales. We also sought to develop systematic profiling data and assays for determining the maturation levels of iN cultures as resources for the community. Shan et al. report methods to generate postnatal-like iPSC-derived neurons at large scale and with long-term stability. They provide extensive characterization data and assays to measure neuronal maturity. They find genes associated with maturation exhibit diverse functions. Their data support the utility of these methods to enable modeling of neurological disorders. View Publication

SummaryHeterotaxy is a disorder characterized by severe congenital heart defects (CHDs) and abnormal left-right patterning in other thoracic or abdominal organs. Clinical and research-based genetic testing has previously focused on evaluation of coding variants to identify causes of CHDs,leaving non-coding causes of CHDs largely unknown. Variants in the transcription factor zinc finger of the cerebellum 3 (ZIC3) cause X-linked heterotaxy. We identified an X-linked heterotaxy pedigree without a coding variant in ZIC3. Whole-genome sequencing revealed a deep intronic variant (ZIC3 c.1224+3286A>G) predicted to alter RNA splicing. An in vitro minigene splicing assay confirmed the variant acts as a cryptic splice acceptor. CRISPR-Cas9 served to introduce the ZIC3 c.1224+3286A>G variant into human embryonic stem cells demonstrating pseudoexon inclusion caused by the variant. Surprisingly,Sanger sequencing of the resulting ZIC3 c.1224+3286A>G amplicons revealed several isoforms,many of which bypass the normal coding sequence of the third exon of ZIC3,causing a disruption of a DNA-binding domain and a nuclear localization signal. Short- and long-read mRNA sequencing confirmed these initial results and identified additional splicing patterns. Assessment of four isoforms determined abnormal functions in vitro and in vivo while treatment with a splice-blocking morpholino partially rescued ZIC3. These results demonstrate that pseudoexon inclusion in ZIC3 can cause heterotaxy and provide functional validation of non-coding disease causation. Our results suggest the importance of non-coding variants in heterotaxy and the need for improved methods to identify and classify non-coding variation that may contribute to CHDs. Coding variants in the transcription factor ZIC3 cause X-linked heterotaxy,a laterality defect causing congenital anomalies. Functional genomic analyses of a ZIC3 intronic variant identified in an X-linked heterotaxy pedigree demonstrated pseudoexon inclusion leading to RNA-splicing disruption,highlighting the importance of whole-genome sequencing to identify potential disease-causing variants. View Publication