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The maturation of brain microvascular endothelial cells leads to the formation of a tightly sealed monolayer,known as the blood–brain barrier (BBB). The BBB damage is associated with the pathogenesis of age-related neurodegenerative diseases including vascular cognitive impairment and Alzheimer’s disease. Growing knowledge in the field of epigenetics can enhance the understanding of molecular profile of the BBB and has great potential for the development of novel therapeutic strategies or targets to repair a disrupted BBB. Histone deacetylases (HDACs) inhibitors are epigenetic regulators that can induce acetylation of histones and induce open chromatin conformation,promoting gene expression by enhancing the binding of DNA with transcription factors. We investigated how HDAC inhibition influences the barrier integrity using immortalized human endothelial cells (HCMEC/D3) and the human induced pluripotent stem cell (iPSC)-derived brain vascular endothelial cells. The endothelial cells were treated with or without a novel compound named W2A-16. W2A-16 not only activates Wnt/?-catenin signaling but also functions as a class I HDAC inhibitor. We demonstrated that the administration with W2A-16 sustained barrier properties of the monolayer of endothelial cells,as evidenced by increased trans-endothelial electrical resistance (TEER). The BBB-related genes and protein expression were also increased compared with non-treated controls. Analysis of transcript profiles through RNA-sequencing in hCMEC/D3 cells indicated that W2A-16 potentially enhances BBB integrity by influencing genes associated with the regulation of the extracellular microenvironment. These findings collectively propose that the HDAC inhibition by W2A-16 plays a facilitating role in the formation of the BBB. Pharmacological approaches to inhibit HDAC may be a potential therapeutic strategy to boost and/or restore BBB integrity. 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

BackgroundLiver disease imposes a significant medical burden that persists due to a shortage of liver donors and an incomplete understanding of liver disease progression. Hepatobiliary organoids (HBOs) could provide an in vitro mini-organ model to increase the understanding of the liver and may benefit the development of regenerative medicine.MethodsIn this study,we aimed to establish HBOs with bile duct (BD) structures and mature hepatocytes (MHs) using human chemically induced liver progenitor cells (hCLiPs). hCLiPs were induced in mature cryo-hepatocytes using a small-molecule cocktail of TGF-? inhibitor (A-83-01,A),GSK3 inhibitor (CHIR99021,C),and 10% FBS (FAC). HBOs were then formed by seeding hCLiPs into ultralow attachment plates and culturing them with a combination of small molecules of Rock-inhibitor (Y-27632) and AC (YAC).ResultsThese HBOs exhibited bile canaliculi of MHs connected to BD structures,mimicking bile secretion and transportation functions of the liver. The organoids showed gene expression patterns consistent with both MHs and BD structures,and functional assays confirmed their ability to transport the bile analogs of rhodamine-123 and CLF. Functional patient-specific HBOs were also successfully created from hCLiPs sourced from cirrhotic liver tissues.ConclusionsThis study demonstrated the potential of human HBOs as an efficient model for studying hepatobiliary diseases,drug discovery,and personalized medicine.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03877-z. 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

AbstractBackgroundOXA1L is crucial for mitochondrial protein insertion and assembly into the inner mitochondrial membrane,and its variants have been recently linked to mitochondrial encephalopathy. However,the definitive pathogenic link between OXA1L variants and mitochondrial diseases as well as the underlying pathogenesis remains elusive.MethodsIn this study,we identified bi?allelic variants of c.620G>T,p.(Cys207Phe) and c.1163_1164del,p.(Val388Alafs*15) in OXA1L gene in a mitochondrial myopathy patient using whole exome sequencing. To unravel the genotype–phenotype relationship and underlying pathogenic mechanism between OXA1L variants and mitochondrial diseases,patient?specific human?induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into myotubes,while OXA1L knockout human immortalised skeletal muscle cells (IHSMC) and a conditional skeletal muscle knockout mouse model was generated using clustered regularly interspaced short palindromic repeats/Cas9 genomic editing technology.ResultsBoth patient?specific hiPSC differentiated myotubes and OXA1L knockout IHSMC showed combined mitochondrial respiratory chain defects and oxidative phosphorylation (OXPHOS) impairments. Notably,in OXA1L?knockout IHSMC,transfection of wild?type human OXA1L but not truncated mutant form rescued the respiratory chain defects. Moreover,skeletal muscle conditional Oxa1l knockout mice exhibited OXPHOS deficiencies and skeletal muscle morphofunctional abnormalities,recapitulating the phenotypes of mitochondrial myopathy. Further functional investigations revealed that impaired OXPHOS resulting of OXA1L deficiency led to elevated reactive oxygen species production,which possibly activated the nuclear factor kappa B signalling pathway,triggering cell apoptosis.ConclusionsTogether,our findings reinforce the genotype–phenotype association between OXA1L variations and mitochondrial diseases and further delineate the potential molecular mechanisms of how OXA1L deficiency causes skeletal muscle deficits in mitochondrial myopathy. 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