技术资料

The oral mucosa plays an important role in maintaining oral and systemic health by protecting the body from harmful environmental stimuli and pathogens. Current reconstructed human gingiva models (RhG) serve as valuable testing platforms for safety and efficacy testing of dental materials,however they lack important phenotypic characteristics typical of the gingival epithelium. We aimed to determine whether incorporating induced pluripotent stem cells (iPSCs) into the hydrogel of a cell-line RhG (reconstructed epithelium on fibroblast-populated-hydrogel) would improve its phenotype. Immortalized human gingival fibroblasts were resuspended with and without iPSCs in collagen-fibrin hydrogels and gingival keratinocytes were seeded on top of the hydrogels to construct RhGs. RhGs were cultured at air-liquid interface for 1,2,4 and 6 weeks and extensively characterized by immunohistochemistry. In situ hybridization for X and Y chromosomes was conducted to identify female iPSCs and male fibroblasts in the RhGs. iPSC-RhGs showed increased epithelial thickening,rete ridge formation,increased cell proliferation and normalized expression of differentiation markers (keratins,involucrin,loricrin,SKALP/elafin) compared to standard RhGs,resulting in an epithelial phenotype very similar to the native gingiva. An increase in apoptotic cells was detected in iPSC-RhGs after 1 week air-exposed culture,and no iPSCs were detected in the hydrogels after 2 weeks air-exposed culture. The increase in apoptotic iPSCs after 1 week air-exposed culture correlated with an increase in keratinocyte proliferation responsible for the superior phenotype observed at 2 weeks. View Publication

Liver disease secondary to an inborn or genetic error of metabolism is a rare group of conditions often associated with chronic ill health and reduced survival. Curative treatment is mainly limited to liver transplantation with major long-term risks. Cell therapy is a promising alternative,but current approaches are ineffective. To develop histotripsy,a non-invasive high-intensity ultrasound procedure for liver tissue mechanical ablation,combined with hepatocyte stem cell implantation as a novel method of reversing liver failure from genetic disease. This study assessed the safety and feasibility of this approach in healthy rodents. Under general anaesthesia,adult rats (n = 12) underwent laparotomy and ultrasound histotripsy to the exposed liver. Around 1 million cells were injected into a single histotripsy cavity in each animal under direct vision (n = 10) with two receiving only histotripsy without cell injection. On completion of cell implant,haemostasis was secured,laparotomy incision closed and the animals recovered. Groups of animals were terminated immediately and after 4 h,8 h,24 h,4 days and 7 days. Liver and vital organs were assessed for procedure-related injuries and evidence of viable implanted cells by histology and immunohistochemistry. All animals successfully recovered,and no complication was observed throughout the study. Created cavities were successfully identified in histological analysis of rat. The presence of human cells was verified using anti-human nuclei antibody confirming successful implantation of liver organoids into decellularised cavities. In this feasibility study,we demonstrated suitability of histotripsy to create decellularised cavities in liver parenchyma. In addition,feasibility of direct transplantation of undissociated liver organoids into the created cavities was demonstrated as a potential approach to treat inborn liver disease by creating nodules of healthy cells capable of performing loss metabolic function. Therapeutic efficacy of this approach will be evaluated in an upcoming study. Graphical Abstract View Publication

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

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

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

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

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

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

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

BackgroundTrichloroethylene (TCE) is a ubiquitous pollutant with potential capacity to induce congenital heart disease (CHD). However,the mechanisms underlying TCE-induced CHD are largely unraveled.MethodsWe exposed zebrafish embryos to TCE to investigate its cardiac development toxicity and related response factor through bulk RNA sequencing. We constructed transgenic fluorescent fish and employed the CRISPR/dCas9 system along with single-cell RNA sequencing to identify the genetic cause of TCE-induced CHD.ResultsWe found that early-stage exposure to TCE induced significant cardiac defects characterized by elongated SV-BA distance,thinned myocardium,and attenuated contractility. Gremlin1 encoding gene,grem1a,a putative target showing high expression at the beginning of cardiac development,was sharply down-regulated by TCE. Consistently,grem1a knockdown in zebrafish induced cardiac phenotypes generally like those of the TCE-treated group,accompanying the disarrangement of myofibril structure. Single-cell RNA-seq depicted that mitochondrial respiration in grem1a-repressed cardiomyocytes was greatly enhanced,ultimately leading to a branch from the normal trajectory of myocardial development. Accordingly,in vitro results demonstrated that GREM1 repression increased mitochondrial content,ATP production,mitochondrial reactive oxygen species,mitochondrial membrane potential,and disrupted myofibril expansion in hPSC-CMs.ConclusionsThese results suggested that TCE-induced gremlin1 repression could result in mitochondrial hyperfunction,thereby hampering cardiomyocyte development and causing cardiac defects in zebrafish embryos. This study not only provided a novel insight into the etiology for environmental stressor-caused cardiac development defects,but also offered a potential therapeutic and preventive target for TCE-induced CHD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-025-02314-9. View Publication