技术资料

Efficient tumor T-cell infiltration is crucial for the effectiveness of T-cell-based therapies against solid tumors. Eosinophils play crucial roles in recruiting T cells in solid tumors. Our group has previously generated induced eosinophils (iEOs) from human pluripotent stem cells and exhibited synergistic efficacy with CAR-T cells in solid tumor inhibition. However,administrated eosinophils might influx into inflammatory lungs,posing a potential safety risk. Mitigating the safety concern and enhancing efficacy is a promising development direction for further application of eosinophils.MethodsWe developed a new approach to generate eosinophils with enhanced potency from human chemically reprogrammed induced pluripotent stem cells (hCiPSCs) with the Toll-like receptor (TLR) 7/8 signaling agonist R848.ResultsR848-activated iEOs (R-iEOs) showed significantly decreased influx to the inflamed lungs,indicating a lower risk of causing airway disorders. Furthermore,these R-iEOs had enhanced anti-tumor functions,preferably accumulated at tumor sites,and further increased T-cell infiltration. The combination of R-iEOs and CAR-T cells suppressed tumor growth in mice. Moreover,the chemo-trafficking signaling increased in R-iEOs,which may contribute to the decreased lung influx of R-iEOs and the increased tumor recruitment of T cells.ConclusionOur study provides a novel approach to alleviate the potential safety concerns associated with eosinophils while increasing T-cell infiltration in solid tumors. This finding offers a prospective strategy for incorporating eosinophils to improve CAR-T-cell immunotherapy for solid tumors in the future. View Publication

Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol,we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination-mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array,efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase,permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences,no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects. Key features • A single-vector system to deliver Cpf1 and crRNA enables the sorting of transfected cells • Efficient and simultaneous multi-modular genome editing exemplified by mutation of MAFB and knockin of AAVS1 loci in a single experiment • Edited PSCs showed minimal off-target effects and can be differentiated into multiple cell types. View Publication

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by sodium channel dysfunction. However,the clinical management of ARVC remains challenging. Identifying novel compounds for the treatment of ARVC is crucial for advancing drug development.PurposeIn this study,we aim to identify novel compounds for treating ARVC.MethodsMachine learning (ML) models were constructed using proteins analyzed from the scRNA-seq data of ARVC rats and their corresponding protein-protein interaction (PPI) network to predict binding affinity (BA). To validate these predictions,a series of experiments in cardiac organoids were conducted,including Western blotting,ELISA,MEA,and Masson staining to assess the effects of these compounds.ResultsWe first discovered and identified SCN5A as the most significantly affected sodium channel protein in ARVC. ML models predicted that Kaempferol binds to SCN5A with high affinity. In vitro experiments further confirmed that Kaempferol exerted therapeutic effects in ARVC.ConclusionThis study presents a novel approach for identifying potential compounds to treat ARVC. By integrating ML modeling with organoid validation,our platform provides valuable support in addressing the public health challenges posed by ARVC,with broad application prospects. Kaempferol shows promise as a lead compound for ARVC treatment. View Publication

Quantum dots,which won the Nobel Prize in Chemistry,have recently gained significant attention in precision medicine due to their unique properties,such as size-tunable emission,high photostability,efficient light absorption,and vibrant luminescence. Consequently,there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors,biomolecular imaging probes,and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs,which often require expensive and toxic precursors,as they offer several merits in eco-friendly synthesis,preparation from renewable sources,and cost-effective production. In this study,we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines,including human dermal fibroblasts,HeLa,cardiomyocytes,induced pluripotent stem cells,and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells,as they were taken up more by cells treated with tumor necrosis factor-? than untreated cells. In addition,our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents,which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases. View Publication

Human embryonic stem cells (hESCs) serve as a valuable in vitro model for studying early human developmental processes due to their ability to differentiate into all three germ layers. Here,we present a comprehensive multi-omics dataset generated by differentiating hESCs into cardiomyocytes via the mesodermal lineage,collecting samples at 10 distinct time points. We measured mRNA levels by mRNA sequencing (mRNA-seq),translation levels by ribosome profiling (Ribo-seq),and protein levels by quantitative mass spectrometry-based proteomics. Technical validation confirmed high quality and reproducibility across all datasets,with strong correlations between replicates. This extensive dataset provides critical insights into the complex regulatory mechanisms of cardiomyocyte differentiation and serves as a valuable resource for the research community,aiding in the exploration of mammalian development and gene regulation. View Publication

Human induced pluripotent stem cell-derived sensory neuron (iPSC-dSN) models are a valuable resource for the study of neurotoxicity but are affected by poor replicability and reproducibility,often due to a lack of optimization. Here,we identify experimental factors related to culture conditions that substantially impact cellular drug response in vitro and determine optimal conditions for improved replicability and reproducibility. Treatment duration and cell seeding density were both found to be significant factors,while cell line differences also contributed to variation. A replicable dose–response in viability was demonstrated after 48-h exposure to docetaxel or paclitaxel. Additionally,a replicable dose-dependent reduction in neurite outgrowth was demonstrated,demonstrating the applicability of the model for the examination of additional phenotypes. Overall,we have established an optimized iPSC-dSN model for the study of taxane-induced neurotoxicity. View Publication

The rare,accelerated aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is commonly caused by a de novo c.1824 C?>?T point mutation of the LMNA gene that results in the protein progerin. The primary cause of death is a heart attack or stroke arising from atherosclerosis. A characteristic feature of HGPS arteries is loss of smooth muscle cells. An adenine base editor (ABE7.10max) corrected the point mutation and produced significant improvement in HGPS mouse lifespan,vascular smooth muscle cell density,and adventitial fibrosis. To assess whether base editing correction of human HGPS tissue engineered blood vessels (TEBVs) prevents the HGPS vascular phenotype and to identify the minimum fraction of edited smooth muscle cells needed to effect such changes,we transduced HGPS iPSCs with lentivirus containing ABE7.10max. Endothelial cells (viECs) and smooth muscle cells (viSMCs) obtained by differentiation of edited HGPS iPSCs did not express progerin and had double-stranded DNA breaks and reactive oxygen species at the same levels as healthy viSMCs and viECs. Editing HGPSviECs restored a normal response to shear stress. Normal vasodilation and viSMC density were restored in TEBVs made with edited cells. When TEBVs were prepared with at least 50% edited smooth muscle cells,viSMC proliferation and myosin heavy chain levels significantly improved. Sequencing of TEBV cells after perfusion indicated an enrichment of edited cells after 5?weeks of perfusion when they comprised 50% of the initial number of cells in the TEBVs. Thus,base editing correction of a fraction of HGPS vascular cells improves human TEBV phenotype. View Publication

The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs),whose role in antiviral immunity is elusive. Here,we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state,GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9,TUG). Following RNA virus infection,GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs,preventing them from activating IFN-? responses. UBXN9 deletion prompts constitutive GLUT4 trafficking,sequestration of RLRs,and attenuation of antiviral immunity,whereas GLUT4 deletion heightens RLR signaling. Notably,reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall,our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs. View Publication

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system,guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study,we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR,immunofluorescence,and Western blot analysis. Additionally,we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes,including CYP450,urea cycle enzymes,and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes,evident as early as 1 day post-differentiation. Interestingly,HLSCs transiently upregulated stem cell markers during differentiation,followed by downregulation after 7 days. Furthermore,differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h,with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system,its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications. View Publication

ABSTRACTThe ultimate aim of nuclear reprogramming is to provide stem cells or differentiated cells from unrelated cell types as a cell source for regenerative medicine. A popular route towards this is transcription factor induction,and an alternative way is an original procedure of transplanting a single somatic cell nucleus to an unfertilized egg. A third route is to transplant hundreds of cell nuclei into the germinal vesicle (GV) of a non-dividing Amphibian meiotic oocyte,which leads to the activation of silent genes in 24 h and robustly induces a totipotency-like state in almost all transplanted cells. We apply this third route for potential therapeutic use and describe a procedure by which the differentiated states of cells can be reversed so that totipotency and pluripotency gene expression are regained. Differentiated cells are exposed to GV extracts and are reprogrammed to form embryoid bodies,which shows the maintenance of stemness and could be induced to follow new directions of differentiation. We conclude that much of the reprogramming effect of eggs is already present in meiotic oocytes and does not require cell division or selection of dividing cells. Reprogrammed cells by oocytes could serve as replacements for defective adult cells in humans. Summary: Stem cell therapy has shed light on incurable diseases. We describe a novel method for cell reprogramming and provide personalized stem cell sources for stem cell therapies. View Publication