技术资料

Dysregulation of mitochondrial function has been implicated in Parkinson’s disease (PD),but the role of mitochondrial metabolism in disease pathogenesis remains to be elucidated. Using an unbiased metabolomic analysis of purified mitochondria,we identified alterations in ?-ketoglutarate dehydrogenase (KGDH) pathway upon loss of PD-linked CHCHD2 protein. KGDH,a rate-limiting enzyme complex in the tricarboxylic acid cycle,was decreased in CHCHD2-deficient male mouse brains and human dopaminergic neurons. This deficiency of KGDH led to elevated ?-ketoglutarate and increased lipid peroxidation. Treatment of CHCHD2-deficient dopaminergic neurons with lipoic acid,a KGDH cofactor and antioxidant agent,resulted in decreased levels of lipid peroxidation and phosphorylated ?-synuclein. CHCHD10,a close homolog of CHCHD2 that is primarily linked to amyotrophic lateral sclerosis/frontotemporal dementia,did not affect the KGDH pathway or lipid peroxidation. Together,these results identify KGDH metabolic pathway as a targetable mitochondrial mechanism for correction of increased lipid peroxidation and ?-synuclein in Parkinson’s disease. An unbiased metabolomic analysis identifies ?-ketoglutarate dehydrogenase metabolic pathway as a targetable mitochondrial mechanism for correction of increased lipid peroxidation in CHCHD2-linked Parkinson’s disease models. View Publication

Human pluripotent stem cells (hPSCs) and their differentiated derivatives represent valuable tools for studying development,modeling diseases,and advancing cell therapy. Recent improvements in genome engineering allow for precise modifications of hPSCs,further enhancing their utility in basic and translational research. Here we describe a Recombinase-Mediated Cassette Exchange (RMCE) platform in hPSCs that allows for the highly efficient,rapid,and specific integration of transgenes. The RCME-mediated DNA integration process is nearly 100% efficient,without negatively affecting the pluripotency or karyotypic stability of hPSCs. Taking advantage of this convenient system,we first established a dual inducible expression system based on the Tet-On and Cumate-On systems,allowing for the inducible expression of two transgenes independently. Secondly,we incorporated a Tet-on inducible system,driving the expression of three genes simultaneously. However,two genes also contain independent degron sequences,allowing for precise control over the expression of each gene individually. We demonstrated the utility of these systems in hPSCs,as well as their functionality after differentiation into cells that were representative of the three germ layers. Lastly,we used the triple inducible system to investigate the lineage commitment induced by the pancreatic transcription factors NKX6.1 and PDX1. We found that controlled dual expression,but not individual expression,biases hPSC embryoid body differentiation towards the pancreatic lineage by inducing the expression of the NeuroD program. In sum,we describe a novel genetic engineering platform that allows for the efficient and fast integration of any desired transgene(s) in hPSCs using RMCE. We anticipate that the ability to modulate the expression of three transgenes simultaneously will further accelerate discoveries using stem cell technology. View Publication

BackgroundMale factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However,the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here,we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of MFI.MethodsWe reprogrammed human dermal fibroblasts (hDFs) to hiPSCs,induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA,2-AG) by LC/MS,receptors (CB1R,CB2R,TRPV1,GPR55) by qPCR,flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA,CB65,CSP,ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to hSSCs.ResultshiPSCs from hDFs expressed the pluripotency markers OCT4,SOX2,NANOG,SSEA4 and TRA-1-60; and could be differentiated into ID4+,PLZF?+?hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10??10 ??10??9 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs,with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA,2-AG,ACPA,CSP and ML184. The EC50 of CB65 was determined to be 2.092?×?10??8 M for support of pluripotency and preservation of stemness on hiPSCs from 78 h. CB65 stimulation at EC50 also increased the yield of ID4?+?hSSCs,PLZF?+?SSPCs and SCP3?+?spermatocytes from day 10 to 12.ConclusionsWe demonstrated here for the first time that stimulation of CB2R results in an increased yield of hiPSCs and hiPSC-derived hSSCs. CB65 is a potent CB2R agonist that can be used to increase the yield of hiPSC-derived hSSCs offering an alternative source of autologous male germ cells for patients with MFI. Increasing the male germ/stem cell pool by CB65 supplementation could be part of the ART-associated protocols in MFI patients with complete germ cell aplasia.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40659-025-00596-4. View Publication

Background?-catenin,acting as the core effector of canonical Wnt signaling pathway,plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models,the ?-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood,partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study,we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of ?-catenin. Our analysis showed that a truncated ?-catenin lacking both N- and C-terminal domains (?N148C) could robustly rescue the DE formation,whereas hyperactive ?-catenin mutants with S33Y mutation or N-terminal deletion (?N90) had limited ability to induce DE lineage. Notably,the ?N148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak ?-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes,whereas the hyperactive ?-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of ?-catenin through weak transactivation,indicating that the levels of ?-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01279-5. 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

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

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

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

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