Alternative polyadenylation (APA) is a critical posttranscriptional mechanism that generates transcriptomic diversity through the production of mRNA isoforms with distinct 3’ UTRs or coding sequences. Current APA analysis based on single-cell RNA sequencing (scRNA-seq) for establishing cell type-specific APA landscapes primarily rely on Read2 data, which lacks precise cleavage site (CS) information. This limitation restricts their ability to achieve precise de novo mapping of polyadenylation sites (PASs). Here, we present s ingle- c ell P oly A denylation ISO form quantification (scPAISO), a computational pipeline designed for de novo identification of PAS and quantification of PAS isoforms in scRNA-seq data, by leveraging the often-discarded Read1 from 3’ tag-based scRNA-seq protocols. Unlike existing tools, scPAISO directly captures mRNA 3’ end cleavage sites, enabling superior performance in motif enrichment (stronger AAUAAA signal) and peak precision (sharper PAS peaks). Moreover, the smaller peak widths enhance the spatial resolution, enabling more accurate detection of closely spaced PASs in the genome. By integrating Read1 and Read2 data, scPAISO achieves isoform-level quantification with an assignment accuracy exceeding 95%. We demonstrate the robustness of scPAISO in identifying PASs and quantifying APA events across diverse biological contexts, including hematopoiesis, systemic sclerosis (SSc), and mouse tissues. We identified stage-specific 3’ UTR lengthening in hematopoietic progenitors, global 3’ UTR remodeling in SSc and tissue-specific polyadenylation (PA) preference along with RNA-binding proteins in mice. Overall, scPAISO represents a significant advancement in the analysis of APA at single-cell resolution and provides a powerful tool for exploring the regulatory landscape of APA, offering new insights into transcriptome complexity and gene regulation in both health and disease. ### Competing Interest Statement The authors have declared no competing interest.

Infection is a leading cause of high mortality in patients with chronic kidney disease (CKD), in which T cell dysfunction is considered as a typical feature in CKD patients. Although platelets are active participants in immune response, their role in regulating T cell function in CKD patients remains unclear. In this study, we demonstrate that CD8 + T cell senescence may be a major contributor to the immunosuppressed state of end-stage renal disease (ESRD) patients, and that platelets derived from ESRD patients can induce premature CD8 + T cell senescence, potentially through mediating mitochondrial dysfunction. Further investigations reveal that platelet-derived microparticles (PMPs) from ESRD patients promote CD8 + T cell senescence via transferring miR-550a-5p, thereby decreasing PMPCB expression and inducing NDUFS8 cleavage failure. Finally, inhibition of miR-550a-5p and pretreatment with Nicotinamide Mononucleotide is capable of preventing platelet-induced CD8 + T cell senescence in ESRD patients. Collectively, these findings suggest that the ectopic transient expression of platelet-derived miR-550a-5p in CD8 + T cells promotes cellular senescence by regulating the PMPCB-NDUFS8 axis, which can be exploited to treat the ESRD-associated immunosenescence.

Single-cell chromatin accessibility profiles are extremely sparse but reflect continuous developmental trajectories. Most existing methods for dimensionality reduction and trajectory analysis optimize reconstruction error or cluster separation, without encoding temporal continuity in the model or providing metrics tailored to this objective. We introduce iAODE, a variational autoencoder that couples a zero-inflated negative binomial likelihood with a latent Neural ODE, low-weight Kullback-Leibler (KL) regularization, and an interpretable reconstruction bottleneck to learn generative, temporally continuous latent spaces. Around iAODE, we build a standardized AnnData benchmark of 248 single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) and 123 single-cell RNA sequencing (scRNA-seq) datasets and a 20-metric evaluation suite that quantifies latent-space continuity, embedding quality, and clustering-coupling structure. Simulations confirm that the metrics respond smoothly to controlled continuity perturbations, and large-scale benchmarks show that the ODE, low-β, and bottleneck components synergistically improve trajectory structure and robustness over established generative and manifold-learning baselines.

Ionizing radiation-induced intestinal injury (IRIII) reduces survival in nuclear accident victims and compromises the efficacy of abdominal radiotherapy, and current treatment options remain limited. Human defensin 5 (HD5)-derived fragments are endogenous regulators of the gut microbiota, which affects host responses to radiation. However, whether these fragments influence intestinal radiosensitivity or can serve as lead compounds for IRIII therapeutics remains unclear. In this study, we investigated the role of HD5-derived fragments in IRIII and developed AT9(C/G), a potent radioprotective oligopeptide based on the lead fragment AT9. Fecal metagenomic and metabolomic analyses revealed that the oral administration of AT9(C/G) enriches Bifidobacterium pseudolongum and increases lithocholic acid (LCA) levels in the intestine. Both murine and clinical studies demonstrated a negative correlation between IRIII severity and fecal LCA levels. The radioprotective effect of LCA was further validated in both mouse models and human small intestinal organoids. Mechanistically, LCA suppresses ferroptosis in irradiated cells by remodeling lipid metabolism. Specifically, LCA activates Takeda G protein-coupled receptor 5 (TGR5), leading to the upregulation of sterol regulatory element-binding protein 1 (SREBP1), which transcriptionally modulates stearoyl-CoA desaturase 1 (SCD1) to catalyze monounsaturated fatty acid production. Pharmacological inhibition of SCD1 or genetic ablation of G-protein coupled bile acid receptor 1 (Gpbar1, encodes TGR5) attenuates the protective effects of AT9(C/G) in mice. This study establishes that an oligopeptide can modulate gut microbiota-derived LCA to confer intestinal radioprotection, presenting a promising preventive strategy against IRIII.

Hematopoietic stem cells (HSCs) are responsible for replenishing blood cells under stress conditions through increasing proliferation and differentiation. After the hematopoietic function has reconstructed, HSCs must re-enter a quiescent state to avoid their depletion, whereas the underlying mechanisms remain to be elucidated. Here, we show that the translocation of nuclear receptor coactivator 2 (NCOA2) into the nucleus is gradually increased in HSCs during the hematopoietic recovery phase after sub-lethal dose irradiation (IR). Although deletion of NCOA2 only slightly affects the steady state hematopoiesis, its deficiency leads to HSC pool exhaustion and delayed hematopoietic recovery after exposure to IR. Further investigations reveal that loss of NCOA2 decreases the quiescence, survival, and long-term reconstituting ability of HSCs following IR due to increased mitochondria-derived oxidative stress. Mechanistically, NCOA2 promotes the clearance of activated or damaged mitochondria by coactivating FOXO3a-dependent transcription of PINK1, which drives HSCs to return to quiescence after being activated by IR stress. Collectively, our findings demonstrate a critical role of NCOA2 in facilitating the restoration of HSC homeostasis after IR via the FOXO3a-PINK1-mediated mitophagy axis and thus provide an additional strategy to prevent hematopoietic failure induced by IR.

PURPOSE:Radiation-induced skin injury (RISI) is common in both radiation therapy and accidental exposure. An appropriate animal model of RISI is of great significance for understanding its injury mechanism and developing medical countermeasures (MCMs). A reproducible, dose-dependent murine model of RISI was established via localized irradiation of the tail skin with graded doses. MATERIALS AND METHODS:C57BL/6 mice were employed to establish RISI model by irradiating 2 cm section of the mouse tail with 20 Gy, 30 Gy, and 40 Gy of single irradiation with RS2000 Biological Irradiator. Skin injuries were scored with a modified semi-quantitative scale based on Kumar scale. H&E staining, measurement of the thickness of epidermis and dermis, IHC for dopachrome tautomerase (DTC), IF staining for α-smooth muscle actin (α-SMA) and Masson staining were used for histopathological evaluations of RISI. RESULTS:A murine model of RISI was established via graded-dose X-ray irradiation of the tail, followed by comprehensive characterization and evaluation of its phenotypic traits. Dynamic alterations in cutaneous melanin-excessive early deposition and subsequent reduction-triggered by tail irradiation rendered radiation-induced erythema undetectable. To address this issue, we specifically revised the early damage scoring criteria of the RISI scale based on Kumar scale. As radiation dose increased, mice exhibited typical symptoms in the irradiated area, including dry desquamation (20 Gy), moist desquamation (30 Gy), ulcers and necrosis (40 Gy), which mimic the key features of clinical RISI. Further histopathological assessment demonstrated a strong correlation between the scoring system and histological changes. Concurrently, we discovered that in the later stage of this model, the interstitial tissue at irradiated site presented a fibrotic phenotype with good dose dependence. CONCLUSION:In conclusion, this study established an easily operable and highly reproducible tail irradiation model, providing one new platform for in-depth research on the mechanisms and translational applications of RISI.

It has been reported that a close relationship exists between the hematopoietic and skeletal systems, and megakaryocytes (MKs) may play a role in maintaining bone homeostasis. However, the precise role and underlying mechanisms of MKs in osteogenesis, particularly under stress conditions, remain largely unknown. Here we demonstrate that deficiency of MKs significantly impairs bone formation, accompanied by a reduction in the number of leptin receptor positive skeletal stem cells (LepR+ SSCs) in MKs conditionally deleted mice. Further investigations reveal that megakaryocytic TGFβ1 promotes the osteogenic differentiation of LepR+ SSCs following irradiation. Notably, thrombopoietin treatment effectively maintains the number of LepR+ SSCs and stimulates bone formation. Moreover, MKs-derived TGFβ1 facilitates zinc ions influx into LepR+ SSCs by activating Slc39a14, thereby alleviating endoplasmic reticulum stress after irradiation. In addition, the increased intracellular zinc levels inhibit PTP1B expression and activate Stat3 signaling, promoting osteogenic lineage commitment. In conclusion, our findings demonstrate that the megakaryocytic TGFβ1 orchestrates the osteogenesis of LepR+ SSCs following irradiation, offering a potential therapeutic strategy for radiation-induced bone loss. This study explores how certain cells in our bones, called skeletal stem cells (SSCs), help maintain bone health and repair damage. The researchers found that a small group of cells, known as leptin receptor-positive (LepR+) SSCs, are crucial for bone repair. The study focused on how megakaryocytes (MKs), a type of bone marrow cell, support these SSCs. The researchers used mice to study the effects of radiation on bones and how MKs help LepR+ SSCs recover. They discovered that MKs release a protein called TGFβ1, which helps LepR+ SSCs absorb zinc ions. This process reduces stress in the cells and encourages them to become osteoblasts. The study also showed that increasing MKs in the bone marrow can improve bone strength after radiation. In conclusion, MKs play a vital role in bone repair by supporting LepR+ SSCs. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

ABSTRACT:Polyploidization resulting from massive DNA synthesis is crucial for megakaryocyte (MK) maturation; however, the regulatory mechanisms of cell fitness on this special cellular process remain poorly understood. Here, we reveal that glutamine synthetase (GLUL) facilitates thrombocytopoiesis by restricting ammonia accumulation during polyploidization. GLUL is found to be distinctly expressed in platelet-producing MKs and increasingly elevated with the progression of polyploidization, whereas GLUL deficiency impairs MK maturation and platelet production. Mechanistically, GLUL detoxifies ammonia derived from adenosine deaminase acting on RNA 1-mediated double-stranded RNA editing in MKs undergoing polyploidization. Ammonia accumulation is observed in MKs defective in GLUL, leading to lysosomal and mitochondrial damage and even cell death. Fulvotomentoside A is identified as a potential GLUL agonist with the capacity to promote thrombocytopoiesis in mice after radiation and chemotherapy injury. Our findings uncover the biological significance of GLUL in MK maturation and provide a new avenue for regulating thrombocytopoiesis.

Hematopoietic stem cells (HSC) are critical for lifelong blood cell generation. After mutation accumulation and functional disruption, HSC may transform into leukemic stem cells (LSC), leading to malignant hematological disorders. However, both HSC and LSC are highly heterogeneous, which hinders our comprehensive understanding of their biological characteristics and clinical application. Here, we identified multimerin 1 (Mmrn1) as a reliable marker for the most primitive HSC and LSC. We found that Mmrn1 was abundantly present in human and mouse HSC. Interestingly, HSC with high levels of Mmrn1 displayed increased quiescence and regenerative capacity, accompanied by megakaryocytic lineage commitment. Importantly, Mmrn1 deficiency gradually impairs HSC self-renewal under stress of transplantation due to reduced quiescence. Additionally, we noticed that Mmrn1 was specifically upregulated in acute myeloid leukemia (AML) cells, and its overexpression predicted poor patient prognosis. Further investigation revealed that Mmrn1 marked a subset of quiescent LSC responsible for AML initiation and development, and that deletion of Mmrn1 delays AML progression. Collectively, these data broaden our knowledge of stem cell heterogeneity in the context of normal and malignant hematopoiesis and advance the precision diagnosis and therapy of AML in the clinic.

PURPOSE:Estrogens are frequently employed as radioprotective agents in nuclear emergencies, yet their effects on radiation combined injury (RCI) are poorly understood. This study evaluated the impact of nilestiol on radiation-wound combined injury (R-W-CI). MATERIALS AND METHODS:Mouse models included 6.0 and 8.0 Gy total body irradiation (TBI), 2.0% of the total body surface area (TBSA) skin wound trauma, and R-W-CI (6.0 or 8.0 Gy TBI followed by a 2% TBSA wound). Nilestriol was administered 3 and 1 day pre-injury. The outcomes assessed included 30-day survival, weight changes, peripheral blood analysis, wound closure, bone marrow-derived clonogenic activity, flow cytometric analysis of hematopoietic stem and progenitor cells, and splenic extramedullary hematopoiesis. RESULTS:Nilestriol significantly improved 30-day survival rate in mice subjected to 8.0 Gy TBI and enhanced hematopoietic recovery in those exposed to 6.0 Gy TBI. In wound-only models, nilestriol suppressed early inflammatory cytokines and impaired wound closure. For R-W-CI model with 8.0 Gy, nilestriol significantly improved the 30-day survival rate but increased the risk of early mortality. For R-W-CI with 6.0 Gy, nilestriol significantly promoted hematopoietic recovery, but led to a significant delay in wound healing by more pronounced inhibitory effects on the early inflammatory response in wounds of R-W-CI. No gender differences were observed in the effects of nilestriol. Additionally, Splenic extramedullary hematopoiesis was inhibited in nilestriol pretreatment groups. CONCLUSION:These findings suggest that pre-irradiation administration of nilestriol can mitigate R-W-CI effects but requires careful consideration due to potential negative impacts on both systemic and local levels.

Ionizing radiation-induced intestinal injury (IRIII) is a catastrophic disease lack of sufficient medical countermeasures currently. Regulation of the gut microbiota through dietary adjustments is a potential strategy to mitigate IRIII. Time-restricted feeding (TRF) is an emerging behavioral nutrition intervention with pleiotropic health benefits. Whether this dietary pattern influences the pathogenesis of IRIII remains vague. We evaluated the impact of TRF on intestinal radiosensitivity in this study and discovered that only daytime TRF (DTRF), not nighttime TRF, could ameliorate intestinal damage in mice that received a high dose of IR. Faecal metagenomic and metabolomic studies revealed that the intestinal creatine level was increased by approximate 9 times by DTRF, to which the Bifidobacterium pseudolongum enrichment contribute. Further investigations showed that creatine could activate the energy sensor AMP-activated protein kinase in irradiated enterocytes and induce phosphorylation of acetyl-CoA carboxylase, resulting in reduced production of polyunsaturated fatty acids and reduced ferroptosis after IR. The administration of creatine mitigated IRIII and reduced bacteremia and proinflammatory responses. Blockade of creatine import compromised the ferroptosis inhibition and mitigation of DTRF on IRIII. Our study demonstrates a radioprotective dietary mode that can reshape the gut microbiota and increase intestinal creatine, which can suppress IR-induced ferroptosis, thereby providing effective countermeasures for IRIII prevention.

Prenatal inflammation exposure (PIE) is associated with increased prevalence of cardiovascular diseases (CVDs) in offspring, including heart failure and hypertension. In this study, we investigated the molecular mechanisms underlying the prenatal programming of cardiac function. Pregnant mice were injected with poly (I:C) (20 mg/kg, i.p.) on day 10.5 of gestation. Mothers and pubs were fed with high-fat diet during lactation. Cardiac tissues of the offspring were collected for analysis. We found that prenatal poly (I:C) exposure significantly reduced fatty acid metabolism and impaired the homeostasis of energy metabolism in the heart tissues of offspring at the age of 4 weeks. RNA-sequencing analysis of the heart tissues revealed that prenatal poly (I:C) exposure resulted in decreased expression of the fatty acid oxidation-related enzymes and increased expression of glycolysis-related enzymes, enabling rewiring of energy metabolism. High-fat intake during lactation partially ameliorated cardiac fatty acid metabolism in the heart tissues and prevented cardiac dysfunction in offspring mice exposed to prenatal poly (I:C) at the age of 16 weeks. Collectively, abnormal cardiac fatty acid metabolism accounts for the prenatal poly (I:C) exposure-induced cardiac dysfunction, highlighting the potential of dietary interventions to prevent cardiac dysfunction for PIE offspring.

Antimicrobial peptides (AMPs) are attractive candidates to combat antibiotic resistance for their capability to target biomembranes and restrict a wide range of pathogens. It is a daunting challenge to discover novel AMPs due to their sparse distributions in a vast peptide universe, especially for peptides that demonstrate potencies for both bacterial membranes and viral envelopes. Here, we establish a de novo AMP design framework by bridging a deep generative module and a graph-encoding activity regressor. The generative module learns hidden ‘grammars’ of AMP features and produces candidates sequentially pass antimicrobial predictor and antiviral classifiers. We discovered 16 bifunctional AMPs and experimentally validated their abilities to inhibit a spectrum of pathogens in vitro and in animal models. Notably, P076 is a highly potent bactericide with the minimal inhibitory concentration of 0.21 μM against multidrug-resistant Acinetobacter baumannii , while P002 broadly inhibits five enveloped viruses. Our study provides feasible means to uncover the sequences that simultaneously encode antimicrobial and antiviral activities, thus bolstering the function spectra of AMPs to combat a wide range of drug-resistant infections.

BACKGROUND:Single-cell RNA sequencing analysis faces critical challenges including high dimensionality, sparsity, and complex topological relationships between cells. Current methods struggle to simultaneously preserve global structure, model cellular dynamics, and handle technical noise effectively. RESULTS:We present GNODEVAE, a novel architecture integrating Graph Attention Networks (GAT), Neural Ordinary Differential Equations (NODE), and Variational Autoencoders (VAE) for comprehensive single-cell analysis. Through systematic evaluation across 10 graph convolutional layers, GAT demonstrated optimal performance, achieving average ARI advantages of 0.108 and 0.112 over alternative graph convolutional layers in VGAE and GNODEVAE architectures respectively, along with ASW advantages of 0.047 and 0.098. Extensive comparison across 50 diverse single cell datasets against 18 existing methods demonstrates that GNODEVAE consistently outperforms three major categories of benchmark methods: 8 machine learning dimensionality reduction techniques, 7 deep generative VAE variants, and 3 graph-based and contrastive learning deep predictive models. GNODEVAE achieved average advantages of 0.112 in reconstruction clustering quality (ARI) and 0.113 in clustering geometry quality (ASW) over standard VGAE, with an average ASW advantage of 0.286 over all benchmark methods in clustering geometry quality. In gene dynamics clustering evaluation, GNODEVAE outperformed Diffusion map and Palantir methods across all geometric metrics. CONCLUSIONS:GNODEVAE establishes a robust computational framework that synergistically combines neighborhood-awareness, dynamic modeling, and probabilistic expressiveness for single-cell multi-omics analysis. The consistent superior performance across diverse datasets demonstrates its effectiveness as a versatile tool for cell clustering, dimensionality reduction, and pseudotime trajectory analysis in both scRNA-seq and scATAC-seq data mining.

The increasing infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) poses a serious threat to global public health. Antimicrobial peptides (AMPs) are alternatives to conventional antibiotics in combating superbugs. However, discovering AMPs with low synthesis costs and strong antibacterial effects against CRAB is challenging. In this study, we synthesized 28 dodecapeptides for bactericidal assessment by site mutation and all-hydrocarbon stapling on the basis of the antibacterial core of human cathelicidin. The linear derivative d12 (Q5RD9I-KR12) and the i, i + 4 stapled peptide d24, which was generated by substituting Val4 and Lys8 of d12 to staples, stood out among the candidates. These short AMPs efficiently bound to bacterial membrane and penetrated it in a lipid A-dependent manner, resulting in low minimal inhibitory concentrations to inactivate CRAB clinical isolates (2.5-20 μg/mL). The CRAB infection mouse models of irradiation-assisted local pulmonary infection and intra-abdominal sepsis revealed that treatment with d12 and d24 significantly eliminated CRAB in vivo and thereby increased mouse survival. Owing to its improved proteolytic resistance, d24 outperformed d12 in suppressing intra-abdominal CRAB infection. The excellent antibacterial effects, good biocompatibility, and facile synthesis make d12 and d24 promising candidates to curb CRAB infections in different application scenarios.

Variational autoencoders (VAEs) serve as essential components in large generative models for extracting latent representations and have gained widespread application in biological domains. Developing VAEs specifically tailored to the unique characteristics of biological data is crucial for advancing future large-scale biological models. Through systematic monitoring of VAE training processes across 31 public single-cell datasets spanning oncological and normal conditions, we discovered that reducing the β value which corresponds to lower disentanglement of VAE significantly improves unsupervised clustering metrics in single-cell data analysis. Based on this finding, we innovatively developed iVAE with an irecon module that, when benchmarked against 8 established dimensionality reduction methods across 5 clustering performance metrics, exhibited superior capabilities in representing single-cell transcriptomic data. The proposed iVAE architecture enhances the interpretability of single-cell data compared to conventional VAE architectures as measured by clustering metrics. Our work establishes a potential foundational VAE architecture for developing specialized large-scale generative models for biological applications.

Redox balance is essential for maintenance of the hematopoietic stem cell (HSC) pool, which ensures the lifelong hematopoiesis. However, oxidative attack induced by various physiopathological stresses always compromises HSC maintenance, while there remains lack of safe and effective antioxidative measures combating these conditions. Here, we show that ferulic acid (FA), a natural antioxidant abundantly present in Angelica sinensis which is a traditional Chinese herb commonly used for promotion of blood production, distinctively and directly promotes HSC maintenance and thereby boosts hematopoiesis at homeostasis, whether supplemented over the long term in vivo or in HSC culture ex vivo. Using a mouse model of acute myelosuppressive injury induced by ionizing radiation, we further reveal that FA supplementation effectively safeguards HSC maintenance and accelerates hematopoietic regeneration after acute myelosuppressive injury. Mechanistically, FA diminishes ferroptosis susceptibility of HSCs through limiting the labile iron pool (LIP), thus favoring HSC maintenance. In addition, the LIP limitation and anti-ferroptosis activity of FA is independent of nuclear-factor erythroid 2-related factor 2 (NRF2), probably relying on its iron-chelating ability. These findings not only uncover a novel pharmacological action and mechanism of FA in promoting HSC maintenance, but also provides a therapeutic rationale for using FA or FA-rich herbs to treat iron overload- and ferroptosis-associated pathologies such as acute myelosuppressive injury.

In living organisms, in situ labeling of norepinephrine (NE) to visualize neuroregulatory mechanisms is of significant importance for the pathological analysis of neurological diseases. Traditional "extraction-detection" methods lack spatiotemporal precision and are disconnected from the dynamic changes of life processes. Fluorescent probes combined with high-resolution microscopy imaging techniques have shown outstanding advantages in biological in situ imaging research. Here, we have developed a NE-specific recognition molecular probe, DQF, by covalently linking the high molar extinction coefficient black-hole quencher BBQ-650 with a fluorescent reporter group, utilizing the fluorescence resonance energy transfer mechanism for prequenching fluorescence. The probe DQF features low self-fluorescence (enhancing the contrast before and after fluorescence activation), long-wavelength emission (658 nm, improving the signal-to-noise ratio), and a sensitive, specific response to NE (16 min, 58.6 μM). Compared to the previously developed probe SXU-NEQ, the self-fluorescence intensity of probe DQF decreased significantly (IDQF/ISXU-NEQ = 35%). Time-dependent density functional theory confirmed the differences in fluorescence quenching mechanisms between the two probes. Confocal cell imaging and flow cytometry experiments confirmed that DQF can sensitively label changes in NE levels in SH-SY5Y neuroblast cells during differentiation. Additionally, probe DQF was applied for imaging NE in the substantia nigra and hippocampus of Parkinson's disease mouse brains, clearly revealing neuronal loss and morphological atrophy, particularly in the compact part of the substantia nigra. Probe DQF is an excellent molecular tool for in situ fluorescence labeling of NE in biological samples, with broad application potential in neurobiological research and the diagnosis of neurological diseases.

Human embryonic stem cell-derived NK (hESC-NK) cells or induced pluripotent stem cell derived NK cells have demonstrated efficacy and safety in clinical trials for cancer therapy and serve as a valuable tool for studying the mechanisms of human NK cell development and effector functions. We previously demonstrated that the methylase METTL3 was essential for the development and effector functions of murine NK cells, but its role in human NK cells remained unknown. Herein, we constructed an H1 ESC strain with reduced METTL3 expression using lentivirus-delivered short hairpin (sh) RNA and generated hESC-NK cells via a two-stage differentiation system. Our findings demonstrated that METTL3 knockdown in hESCs reduced the proportion of hematopoietic stem and progenitor cells (HSPCs, CD34+ cells) during embryoid bodies (EBs) formation, and impaired subsequent differentiation into mature NK cells. Moreover, ESC-NK cells derived from shMETTL3-ESC (called shMETTL3-ESC-NK) showed impaired anti-tumor activity, evidenced by downregulation of mRNA and protein levels of critical effectors (perforin, granzyme B and IFN-γ) and reduced cytotoxicity against target cells. Furthermore, both mRNA and protein levels of T-BET and EOMES were significantly down-regulated in shMETTL3-ESC-NK cells. These transcription factors are critical for NK cell development and cytotoxicity, and their downregulation may underlie the maturation defects of shMETTL3-ESC-NK cells. Collectively, our study elucidates that METTL3 promotes the development, maturation and cytotoxicity of hESC-NK cells, recapitulating previous reports in murine NK cells.