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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

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

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

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

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

Autoimmune uveitis is a leading cause of severe vision loss,and animal models provide unique opportunities for studying its pathogenesis and therapeutic strategies. Here we employ scRNA-seq,RNA-seq and various molecular and cellular approaches to characterize mouse models of classical experimental autoimmune uveitis (EAU),revealing that EAU causes broad retinal neuron degeneration and marker downregulation,and that Müller glia may act as antigen-presenting cells. Moreover,EAU immune response is primarily driven by Th1 cells,and results in dramatic upregulation of CC chemokines,especially CCL5,in the EAU retina. Accordingly,overexpression of CCR5,a CCL5 receptor,in mesenchymal stem cells (MSCs) enhances their homing capacity and improves their immunomodulatory outcomes in preventing EAU,by reducing infiltrating T cells and activated microglia and suppressing Nlrp3 inflammasome activation. Taken together,our data not only provide valuable insights into the molecular characteristics of EAU but also open an avenue for innovative MSC-based therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03134-3. View Publication

SummaryInducible loss-of-function strategies are crucial for understanding gene function. However,creating inducible,multiple-gene knockout models is challenging and time-consuming. Here,we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs,designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid,and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones.For complete details on the use and execution of this protocol,please refer to Matsui et al.1 Graphical abstract Highlights•Dox-inducible CRISPRi system to silence multiple genes concurrently•Instructions for generating CRISPRi hPSCs transduced with four sgRNAs•FOXA1/A2/A3-CRISPRi system represses expression of all three FOXA genes by 95% Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Inducible loss-of-function strategies are crucial for understanding gene function. However,creating inducible,multiple-gene knockout models is challenging and time-consuming. Here,we present a protocol for establishing a doxycycline-inducible CRISPR interference (CRISPRi) system to concurrently silence multiple genes in human induced pluripotent stem cells (hPSCs). We describe the steps for establishing host CRISPRi hPSCs,designing and cloning single-guide RNAs (sgRNAs) into a lentivirus plasmid,and establishing monoclonal CRISPRi hPSC lines transduced with sgRNAs. We also detail the procedures for selecting effective CRISPRi clones. View Publication

Background/Objectives: The limited translatability of preclinical experimental findings to patients remains an obstacle for successful treatment of brain diseases. Relevant models to elucidate mechanisms behind brain pathogenesis,including cell-specific contributions and cell-cell interactions,and support successful targeting and prediction of drug responses in humans are urgently needed,given the species differences in brain and blood-brain barrier (BBB) functions. Human microphysiological systems (MPS),such as Organ-Chips,are emerging as a promising approach to address these challenges. Here,we examined and advanced a Brain-Chip that recapitulates aspects of the human cortical parenchyma and the BBB in one model. Methods: We utilized human primary astrocytes and pericytes,human induced pluripotent stem cell (hiPSC)-derived cortical neurons,and hiPSC-derived brain microvascular endothelial-like cells and included for the first time on-chip hiPSC-derived microglia. Results: Using Tumor necrosis factor alpha (TNF?) to emulate neuroinflammation,we demonstrate that our model recapitulates in vivo-relevant responses. Importantly,we show microglia-derived responses,highlighting the Brain-Chip’s sensitivity to capture cell-specific contributions in human disease-associated pathology. We then tested BBB crossing of human transferrin receptor antibodies and conjugated adeno-associated viruses. We demonstrate successful in vitro/in vivo correlation in identifying crossing differences,underscoring the model’s capacity as a screening platform for BBB crossing therapeutic strategies and ability to predict in vivo responses. Conclusions: These findings highlight the potential of the Brain-Chip as a reliable and time-efficient model to support therapeutic development and provide mechanistic insights into brain diseases,adding to the growing evidence supporting the value of MPS in translational research and drug discovery. View Publication

Neutrophils are essential innate immune cells with unusual anti-microbial properties while dysfunctions of neutrophils lead to severe health problems such as lethal infections. Generation of neutrophils from human induced pluripotent stem cells (hiPSCs) is highly promising to produce off-the-shelf neutrophils for transfusion therapies. However,the anti-microbial potencies of hiPSCs derived neutrophils (iNEUs) remain less documented. Here,we develop a scalable approach to generate iNEUs in a chemical defined condition. iNEUs display typical neutrophil characters in terms of phagocytosis,migration,formation of neutrophil extracellular traps (NETs),etc. Importantly,iNEUs display a strong killing potency against various bacteria such as K.pneumoniae,P.aeruginosa,E.coli and S.aureus. Moreover,transfusions of iNEUs in mice with neutrophil dysfunction largely enhance their survival in lethal infection of different bacteria. Together,our data show that hiPSCs derived neutrophils hold strong anti-microbial potencies to protect severe infections under neutrophil dysfunction conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00227-z. View Publication

IntroductionAccumulating evidence indicates that the increased presence of astrocytes is fundamentally linked to the neurological dysfunctions observed in individuals with Down syndrome (DS). REST (RE1-silencing transcription factor),as a chromatin modifier,regulates 15,450 genes in humans. REST is a key regulatory element that governs astrocyte differentiation,development,and the maintenance of their physiological functions. The downregulation of REST may disrupt the homeostatic balance of astrocytes in DS.MethodsThis study aims to elucidate the role of REST in DS-astrocytes through comprehensive transcriptomic analysis and experimental validation.ResultsTranscriptomic analysis identified that REST-targeted differentially expressed genes (DEGs) in DS astrocytes are enriched in pathways associated with inflammatory response. Notably,our findings in astrocytes derived from DS human induced pluripotent stem cells (hiPSCs) show that the loss of nucleus REST leads to an upregulation of inflammatory mediators and markers indicative of the presence of reactive astrocytes. Lithium treatment,which restored nucleus REST in trisomic astrocytes,significantly suppressed the expression of these inflammatory mediators and reactive astrocyte markers.DiscussionThese findings suggest that REST is pivotal in modulating astrocyte functionality and reactivity in DS. The loss of REST in DS-astrocytes prompts the formation of reactive astrocytes,thereby compromising central nervous system homeostasis. Lithium treatment possesses the potential to rescue astrocyte reactivity in DS by restoring nucleus REST expression. View Publication