Abstract Background Argininosuccinate synthase (ASS1) is a pivotal enzyme involved in the urea cycle, playing a crucial role in aspartate catabolism, arginine and nitric oxide biosynthesis. These biological processes are crucial for the growth and development of mammals. However, the functions of urea cycle-related genes in mouse embryonic stem cells (mESCs) remain largely unclear. Here, we investigated the impact of ASS1 knockout on the mESCs pluripotency and its role in determining cell fate. Methods ASS1 was knocked out in mESCs using CRISPR-Cas9. Changes in pluripotency post-knockout were analyzed via immunofluorescence, Western blotting, differentiation assays, and chimera formation. Cardiomyocyte differentiation assays evaluated the impact on cardiac lineage specification. RNA sequencing (RNA-seq), Western blotting, and signaling pathway inhibitors were used to investigate alterations in molecular signatures and regulatory mechanisms. Results ASS1 knockout did not compromise mESCs pluripotency maintenance or self-renewal but perturbed the cell cycle. It upregulated mesendoderm markers while downregulating ectoderm markers. Notably, ASS1 deficiency enhanced cardiomyocyte differentiation potential. The multi-lineage differentiation markers were reversed by either ASS1 overexpression or treatment with Wnt/β-catenin inhibitors. Conclusions ASS1 knockout directs mESCs toward mesendoderm lineage commitment, thereby promoting beating cardiomyocyte generation. Thus, ASS1 acts as a regulatory switch whose absence drives mesendoderm differentiation and enhances cardiomyocyte production. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-025-04622-w. Keywords: Argininosuccinate synthase (ASS1), Mouse embryonic stem cells (mESCs), Mesendoderm, Cardiomyocyte, Wnt/β-catenin Introduction The urea cycle (UC) is a four-step enzymatic process that converts nitrogen derived from ammonia and aspartate into urea. As the primary detoxification pathway for ammonia, impairments at any enzymatic step can lead to disease [[38]1]. Consequently, the expression of UC enzymes and their metabolites dynamically changes in response to physiological and pathological conditions, making them crucial for cellular proliferation, survival, and growth. This cycle involves several transporters and catalytic enzymes. Argininosuccinate synthase (ASS1) and Argininosuccinate lyase (ASL) in the urea cycle are located in the cytoplasm and catalyze the synthesis of arginine and fumarate from citrulline and aspartate [[39]1, [40]2]. Pluripotent stem cells (PSCs) are characterized by their capacity to differentiate into cells of all three germ layers, as well as their ability to sustain self-renewal [[41]3]. Mouse embryonic stem cell (mESC) lines were initially derived from the inner cell mass (ICM) of mouse blastocysts [[42]4]. Owing to their exceptional self-renewal capacity and ability to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm), mESCs offer significant potential for diverse research fields and clinical applications. In addition to growth factors and extracellular matrix signaling, metabolic pathways also provide important signals for self-renewal and differentiation potential of stem cells [[43]5]. Notably, multiple studies indicate that additional amino acids, including glutamine, methionine, arginine, proline and threonine, have important roles in pluripotency maintenance and proliferation of PSCs [[44]6–[45]10]. Arginine, an intermediate metabolite in the UC pathway, also functions as a semi-essential amino acid that can be imported into cells via membrane transporters. Consequently, normal cells do not completely depend on external arginine. However, cells with downregulated ASS1 expression often rely on exogenous arginine to sustain intracellular pools. Arginine and its metabolic derivatives—including nitric oxide, polyamines, and nucleotides—are indispensable for cellular physiology. Disruptions in urea cycle activity can profoundly impair cellular ammonia metabolism and polyamine biosynthetic capacity [[46]11]. In general, human and mouse cells possess the capacity to intracellularly synthesize arginine from citrulline and aspartate by utilizing two rate-limiting enzymes ASS1 and ASL [[47]12]. Mutations in the ASS1 gene result in a pathological change in the urea cycle, leading to elevated levels of blood ammonia and citrulline [[48]13]. The deficiency of ASS1 causes Citrullinemia type I (CTLN1), an autosomal recessive urea cycle disorder [[49]14, [50]15]. Citrullinemia mouse established early on exhibited severe hyperammonemia, elevated citrulline levels in the blood, and died within 1–2 days after birth, closely resembling the human disease [[51]16]. Hepatocyte-like cells (HLCs) derived from CTLN1 patient induced pluripotent stem cells (iPSCs) recapitulate this phenotype, where L-arginine supplementation enhances ammonia clearance and ureagenesis [[52]17]. Cancer studies reveal that ASS1 deficiency increases aspartate availability for pyrimidine synthesis, promoting proliferation [[53]18]. Notably, these phenotypes extend beyond ASS1 itself, as its key substrate—arginine—plays multiple physiological roles that may contribute to the observed phenotypes. For example, arginine not only acts as a metabolite catalyzed by ASS1 in urea cycle but also the exclusive substrate for nitric oxide synthase to generate nitric oxide, which acts as a diffusible intercellular messenger. Herring et al. reported that arginine can enhance placental blood flow, stimulate placental angiogenesis to grow, and facilitate the transfer of nutrients from the mother to the fetus [[54]19]. In early pregnancy, arginine depletion resulted in blastocyst reversible developmental arrest by regulating trophectoderm cell outgrowth [[55]20]. These effects following arginine change were potentially attributed to the nitric oxide production. Nevertheless, arginine is also implicated in the metabolism of polyamines, aspartate, and various cell signaling pathways [[56]21–[57]26]. These results suggest that arginine content of the blastocyst environment could be an important factor in the control of embryo implantation, embryonic/fetal survival, growth, and development. Furthermore, arginine levels are correlated with histone acetyltransferases (HATs/KATs), thus it may be involved in global histone acetylation. The global acetylation level of cells is known to be intricately linked with numerous signaling pathways and transcription factors [[58]27, [59]28]. In cancer models, arginine deprivation impairs mitochondrial function, reducing metabolites like α-ketoglutarate (α-KG). As α-KG is an essential cofactor for JmjC-domain histone demethylases (JMJDs) and TET DNA demethylases, its decline elevates histone methylation during arginine scarcity [[60]29–[61]32]. Although arginine-mediated epigenetic regulation is well-documented, how ASS1 deficiency alters arginine metabolism to influence cellular differentiation in diverse cell types remains poorly understood. Notably, knockout effects of the arginine synthesis gene ASS1 have been poorly reported in mESCs. Here, we demonstrate that ASS1 deficiency promotes mesendoderm lineage commitment and cardiomyogenesis by dysregulating Wnt/β-catenin signaling. Materials and methods Cell culture The W2 mESCs line was established from E3.5 blastocysts harvested from Oct4-ΔPE-GFP (GOF/GFP, mixed background of MF1, 129/sv, and C57BL/6 J strains) × 129/sv F1 mouse [[62]33]. Control mESCs (W2) and ASS1-knockout mESCs (A26 and A27) were cultured under feeder-free conditions on fibronectin (1 mg/mL in PBS, Millipore) as attachment factor in 2i/LIF culture medium: PD0325901 (1 μM, Miltenyi Biotec), CHIR99021 (3 μM, Miltenyi Biotec) and leukemia inhibitory factor (1000 U/mL, Millipore) added into basic N2B27 medium including 50% DMEM/F12 (Gibco), 50% Neurobasal (Gibco), 1 × non-essential amino acids (NEAA, Gibco), 2 mM GlutaMax (Gibco), 1 × Penicillin/Streptomycin (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1% B27 (Gibco), 0.5% N2 (Gibco) supplemented with 50 mg/L BSA (Gibco). The mESCs usually are passaged regularly at every 2–3 days using StemPro Accutase (Life technology) as a cell dissociation agent. For Wnt/β-catenin pathway modulation, control group W2 was cultured in N2B27 medium supplemented with 2i/LIF. The experimental group was treated with 2i/LIF (A27) or LIF (1000 U/mL, Millipore) plus PD0325901 (1 μM, Miltenyi Biotec) (A27-LP) or LIF (1000 U/mL, Millipore) plus PD0325901 (1 μM, Miltenyi Biotec) plus XAV939(5 μM, MedChemExpress) (A27-LPX) on the basis of N2B27 medium. Derivation of XEN and EpiSCs cell lines To derive the extra-embryonic endoderm (XEN) cell lines, a previous protocol was employed [[63]34]. Embryos at the E3.5 stage with a CD1 (ICR) background were placed in Tyrode’s solution (Sigma-Aldrich) to remove the zona pellucida. They were then washed with M2 (Sigma-Aldrich) and transferred into XEN cell derivation medium, which consisted of 30% TS medium and 70% Feeder-conditioned medium from mouse embryonic fibroblasts, without any growth factors. The TS medium contained RPMI 1640 (Gibco), 20%FBS (Gibco), 1% Penicillin/Streptomycin (Gibco), 1 mM Sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco), and 100 µM β-mercaptoethanol (Gibco). The plates were pre-treated with fibronectin before use. After 3–5 days of culture, the blastocysts attached and formed an outgrowth. Between days 9–12, the spreading outgrowth was disaggregated into single cells using TrypLE (Gibco) for 7 min, and the dissociation was stopped by using XEN cell derivation medium. The cell pellets were resuspended and seeded onto 24-well plates that had been pre-treated with fibronectin. After 2–3 passages at high confluence, the XEN cells can be used for RNA extraction. To convert mESCs to epiblast stem cells (EpiSCs), 1 × 10^5 W2 mESCs were seeded onto fibronectin-coated 6-well plates and cultured with 2i/LIF. The 2i/LIF medium of the cells was withdrawn one day later and the cells were re-cultured in N2B27 medium supplemented with Activin-A (20 ng/mL) and FGF2 (12 ng/mL), and cells were harvested and RNA extracted two days later. All cells were cultured in a regular cell culture incubator (Thermo Fisher Scientific) with ambient O[2] and 5% CO[2] at 37 °C. Plasmid construction and transfection and establishment of ASS1-knockout mESCs ASS1-knockout mESCs were generated by CRISPR/Cas9, and pairs of sgRNA were used to knockout the target exons. The two stable ASS1-knockout cell lines derived from W2 mESCs were named A26 and A27, respectively. The single guide RNA (sgRNA) design tool created by Dr. Zhang’s laboratory ([64]http://zlab.bio/guidedesign-resources) was employed to design sgRNAs for the clustered regularly interspaced short palindromic repeats CRISPR/Cas9 system, with the purpose of targeting the ASS1 gene in the mouse genome. SgRNA sequences are listed in Supplementary Table [65]1. The sgRNA was inserted into the pSpCas9 (BB)-2A-Puro (PX459) V2.0 plasmid, which had been linearized using the BbsI restriction enzyme (Thermo Fisher Scientific). The Cas9 and sgRNA were coexpressed in the pSpCas9 (BB)-2A-Puro (PX459) V2.0 plasmid. Subsequently, the plasmids containing the sgRNA were transfected into mESCs using Lipofectamine 3000 (Invitrogen). The transfected cells were then exposed to 0.6 μg/μL of puromycin 48 hours (h) after transfection to select for successfully transfected cells. The selected cells were then plated onto fibronectin-coated 96-well plates to isolate single cell colonies. Approximately 15 days after colony formation, individual colonies were picked and expanded. Genomic DNA was extracted from these colonies using the TIANamp Genomic DNA Kit (TIANGEN Biotech) and subsequently the target fragment was amplified and verified by Sanger sequencing. The cDNA of ASS1 gene was cloned by polymerase chain reaction (PCR) with PrimeSTAR GXL DNA Polymerase (Takara), and the product was purified by AxyPrep PCR cleanup kit (Axygen) according to the manufacturer’s instructions. The PCR products and vector fragments were then digested simultaneously with Nhel (Takara) and Notl (Takara) and ligated to the pCMV-Neomycin plasmid. Correct plasmids were confirmed by DNA sequencing. The transfections were performed using an Amaxa Nucleofector machine (Lonza) according to the manufacturer’s protocol‚ with 2 μg DNA (pCMV-Neomycin) or 2 μg DNA (pCMV-ASS1-Neomycin). RNA was extracted 48 h after transfection and was reverse transcribed for qPCR according to the manufacturer’s protocol. RNA extraction and reverse transcription‑quantitative real‑time PCR (RT‑qPCR) Total RNA was obtained from cultured cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was then synthesized using a Reverse Transcription System (Promega), followed by real-time quantitative polymerase chain reaction (RT-qPCR) using the GoTaq® qPCR Master Mix (Promega) on a LightCycler 96 Instrument II (Roche). Each experiment was repeated in triplicate, and the specific primer pairs used can be found in Supplementary Table [66]1. Western blot To extract proteins, 6 × 10^6 cells were lysed in buffer containing 137 mM NaCl, 20 mM Tris (pH 8.0), 100 g/L glycerol, 4 g/L EDTA, 50 g/L Triton X-100, 1 μL/mL PMSF (0.1 M) and 10 μL/mL phosphatase inhibitor (10 g/L). The cell lysate was placed on ice for 15 min and centrifuged at 12,000 rpm for 10 min at 4 °C, the supernatant was collected and the concentration measured by BCA Protein Assay Kit (Thermo Scientific). The remaining cell lysates were then added with SDS-PAGE sample loading buffer (Beyotime) according to the manufacturer’s instructions. The denatured proteins underwent electrophoresis on a 12% Bis–Tris gels and then were transferred to a polyvinylidene difluoride (PVDF) membrane using electrophoretic blotting. Membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with peroxidase- conjugated secondary antibodies for 1 h, and signals were detected using Enhanced chemiluminescence (ECL) (Thermo Scientific). Proteins from the cytoplasm and nucleus were extracted using Minute Cytoplasmic and Nuclear Extraction Kits for Cells (Invent Biotechnologies). Immunofluorescence (IF) staining The cells for immunofluorescence assays were first treated with 4% paraformaldehyde (PFA) for 30 min at room temperature to fix, and then blocked and permeabilized with a solution of 1% BSA in PBS containing 0.1% Triton X-100 (Sigma). Following the blocking and permeabilization, the cells were incubated with the primary antibody at 4 °C overnight. The cells were then washed three times in 1% BSA, 0.1% Triton X-100 in PBS for 5 min per wash, and subsequently incubated with a secondary antibody for 1 h at room temperature in the dark. After this, the cells were washed once for 5 min in 1% BSA (Gibco), 0.1% Triton X-100 in PBS and twice for 5 min in PBS. Nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) staining, and the samples were subsequently observed with a laser confocal microscope (NIKON A1R). AP staining Alkaline phosphatase staining was conducted using the Alkaline Phosphatase Staining Kit (Yeasen), following the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde (PFA) and washed with PBS before staining for 10 min. After staining, cells were washed with PBS and photographed under a microscope. Flow cytometry Cells were dissociated using Accutase and passed through a 70-μm cell strainer (BD Biosciences) to generate a single-cell suspension. The cells were fixed with 4% paraformaldehyde for 20 min and washed twice in PBS for at least 5 min each. Following incubation in 1% BSA for 30 min, the cells were permeabilized with 0.2% Triton X-100 for 10 min and washed three times in PBS supplemented with 0.05% Triton X-100. Next, the cells were incubated with 647-conjugated primary antibodies (cTnT, 1:200 dilution) for 1 h at room temperature. After PBS washes, the cells were analyzed by flow cytometry using an CytoFLEX flow cytometry (Beckman), and the data were processed with FlowJo 10.4 software. Cell cycle assay The analysis of the cell cycle was carried out following the guidelines provided by the manufacturer using the Cell Cycle and Apoptosis Analysis Kit (Beyotime). In brief, cells were dissociated using Accutase and subsequently gathered through centrifugation. The collected cells were then fixed in 70% ethanol for 2 h. Following fixation, the cell pellet was treated with propidium iodide (PI) staining solution at 37 °C in the dark for 30 min, then strained through a cell filter to remove large cell clumps. Flow cytometry (Beckman) was then performed on the cells, and the resultant data was analyzed utilizing Modfit software. Cell apoptosis assay The apoptosis levels were detected using Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime) in both control and ASS1-knockout group cells at day 5 post-inoculation. Cells in each group were digested with Accutase, centrifuged at 1500 rpm/min for 5 min, 5 × 10^5 mESCs were collected, and carefully washed twice with PBS. According to the manufacturer’s instructions, 500 μL of Binding Buffer was added to resuspend the cells, followed by the addition of 5 μL Annexin V-FITC and 10 μL Propidium Iodide to each tube, with gentle mixing. The samples were incubated at room temperature for 15 min in the dark and then analyzed using a CytoFLEX flow cytometer (Beckman) and CytExpert software (Beckman Coulter Life Sciences, China). N2B27 differentiation For N2B27 differentiation, about 4000 cells were directly seeded in one well of a 48-well plate, and 50 wells in the control mESCs and 50 wells in the ASS1-knockout mESCs were seeded in 48-well plates. After 10 days of culture with basic N2B27 differentiation medium, pictures were taken using microscope, and the number of wells containing beating cells was counted, and finally RNA samples were collected. Animals Female CD1 (ICR) mice (6–8 weeks old) were purchased from Beijing Wei tong Lihua Laboratory Animal Technology Co., Ltd (Beijing, China). All mice were performed during and at the end of the experiment under tribromoethanol (EasyCheck) anaesthesia, and animals were sacrificed using CO[2] inhalation. All animal experiments involved in this study were approved by the Institutional Animal Care and Use Committee at Inner Mongolia University. Generation of chimera For chimaeras, A27 mESCs were usually made to express a CAG–H2B-tdTomato expression cassette by piggyBac transposon transposition. Subsequently, 12–15 cells of A27 mESCs were injected into the cavity of blastocysts on embryonic day 3.5 (E3.5) using a piezo-assisted micromanipulator connected to an inverted microscope. Blastocysts scheduled for microinjection were harvested from ICR strain donor mice. Recipient mice were anesthetized with tribromoethanol (EasyCheck) before embryo transfer. The injected blastocysts were cultured in KSOM medium (Millipore) for 2 h and subsequently transplanted into the uteri of pseudopregnant ICR female mice. On embryonic day 13.5 (E13.5), the mice were euthanized by CO[2] gas overdose inhalation before embryos collecting. No mortality occurred outside of planned euthanasia or humane endpoints. Cardiac differentiation mESCs were submitted to cardiac differentiation according to Chen et al. [[67]35] In brief, the mESCs colonies were trypsinized and dispersed into a single cell suspension in differentiation medium containing (4.5 g/L) Dulbecco’s Modified Eagle’s medium (DMEM; Gibco) supplemented with 20% (v/v) heat-inactivated FBS, 2 mM L-glutamine (Sigma-Aldrich), 50 U/ml penicillin–streptomycin (Gibco), 1% (v/v) nonessential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Gibco) and 1% dimethyl sulfoxide (DMSO; Sigma-Aldrich). Approximately 400 cells in each 20 μL drop were plated on the lids of 100 mm plates (Corning) and cultured for 2 days to form embryoid EBs. These lids were placed back onto 100 mm dishes, containing 10 mL PBS (HyClone), to prevent those hanging drops from drying out. Subsequently, EBs were cultured in suspension for 3 days in 60 mm plates (Corning) using the same differentiation medium. Then, the EBs were transferred to 0.1% gelatin-coated 48-well plates and cultured in same differentiation medium, without DMSO. ASS1-knockout and control groups were seeded to 50 wells in 48-well plate, each well with one EB. Medium was changed every alternate day, and microscopic observations were made to identify the appearance of beating cardiomyocyte clusters in the EBs outgrowth. RNA-seq and analysis W2 mESCs and ASS1-knockout A27 mESCs total RNA was isolated using the RNeasy Mini Kit. cDNA was synthesized from the purified RNA templates and sequenced using the Illumina HiSeq10 × . The raw image data obtained from the sequencing were converted into sequence data, also known as raw data or raw reads. The raw data underwent initial processing to eliminate reads with more than 20% low quality bases and to remove adaptors. Subsequently, the clean data were aligned to the reference genome (Ensembl_release109) using Hisat2 with the default settings. Stringtie was next used to reconstruct transcripts and RSEM was used to calculate the expression of all genes in each sample. After obtaining the tpm values of the genes, subsequent analyses were performed. Differentially expressed genes (DEGs) in different samples were identified by the DEseq2 R package, using a fold change threshold of ≥ 2 and an adjusted p value of ≤ 0.05. Heatmaps of selected genes were generated using OmicShare tools ([68]www.omicshare.com/tools). Additionally, GO and KEGG enrichment analyses were conducted using the clusterProfiler R package, while Gene set enrichment analysis (GSEA) was carried out using the GSEA software developed by the Broad Institute. Transcription factor targeting analysis was performed by using JASPAR database to obtain the motif information of transcription factor binding, and then MEME FIMO software was used to predict transcription target genes. Statistical analysis Graph plots and statistical analyses were conducted using GraphPad Prism 9.0 software. Descriptive statistics were generated for all quantitative data, expressed as mean ± SD, with the values computed from three samples per group and three technical replicates for each sample. Statistical analysis was performed with unpaired two-tailed Student’s t test or two-way ANOVA, which were indicated in the figure legends. The experimental results leading to a p-value < 0.05 were considered statistically significant. Statistically significant p values were indicated in figures as follows: ∗ , p < 0.05; ∗  ∗ , p < 0.01; ∗  ∗  ∗ , p < 0.001. Results ASS1 is dispensable for maintaining stem cell identity in mESCs Tri-lineages of the embryo proper and extraembryonic endoderm stem (XEN) cells are derived from epiblast (Epi) and primitive endoderm (PrE), respectively [[69]4, [70]36, [71]37]. Following embryo implantation, epiblast cells undergo irreversible transcriptional and epigenetic modifications that prevent their reversion to the primed pluripotency state. However, subsequent studies have demonstrated that the leukaemia inhibitory factor (LIF)-STAT3 signaling can reprogram advanced epiblast cells from embryonic day 5.5–7.5 mouse embryos into embryonic stem cell-like cells (rESCs) [[72]38]. Recently, there have been reports on the isolation of primitive endoderm stem cells (PrESCs) in mice. Notably, transcriptomic analysis of PrESCs established by Ohinata et al. demonstrated significant downregulation of ASS1 during PrE commitment [[73]39]. To determine whether ASS1 expression undergoes lineage-specific regulation, we used epiblast stem cells (EpiSCs) and XEN cells, alongside terminally differentiated mouse embryonic fibroblasts (MEFs) as controls. We found that ASS1 transcripts were abundantly expressed in mESCs as compared with differentiated cells such as MEFs, EpiSCs and XEN (Fig. [74]1A). We next investigated whether ASS1 expression was essential for maintaining mESCs' self-renewal state. ASS1 biallelic knockout (at Exon 3) mESCs were successfully generated by the CRISPR/Cas9 gene editing system (Figure [75]S1A). We named the control mESCs as W2 and the two stable ASS1-knockout cell lines as A26 and A27, and we confirmed the depletion of ASS1 protein before collecting the cells for further analysis (Fig. [76]1B). Fig. 1. [77]Fig. 1 [78]Open in a new tab ASS1-knockout mESCs are pluripotent and can self-renew. (A) RT-qPCR analysis of ASS1 mRNA expression in mESCs, EpiSCs, XEN, and MEFs. Values were normalized to β-Actin expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. one-way ANOVA followed by Dunnett’s multiple comparison tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Western blot for ASS1 in control (W2) and ASS1-knockout mESC (A26 and A27). (C) RT-qPCR analysis of arginine transporter gene, including Slc7a3, Slc7a1, Slc7a5 and Slc7a7, in control (W2) and ASS1-knockout mESCs (A26 and A27). Values were normalized to β-Actin expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. P values were calculated by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant. (D) Alkaline phosphatase (AP) staining images of control (W2) and ASS1-knockout mESCs (A26 and A27). Scale bars, 100 μm. (E) Cell proliferation curves in control (W2) and ASS1-knockout mESCs (A26 and A27). Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (F) Representative image of apoptotic cells captured on day 5 of cell culture in control (W2) and ASS1-knockout mESCs (A26 and A27). Four quadrants are indicated: dead cells (upper left), normal cells (lower left), late apoptotic cells (upper right), and early apoptotic cells (lower right). (G) The quantification of apoptosis in control (W2) and ASS1-knockout mESCs (A26 and A27). Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (H) Cell-cycle analysis of control (W2) and ASS1-knockout mESCs (A26 and A27). Representative histograms show distribution of cells in sequential phases (G0/G1, left; S, middle; and G2/M, right) of cell cycle. Bar chart shows the percentage of cell population (10,000 cells per sample). Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test) ASS1 is a rate limiting enzyme for intracellular arginine synthesis, and the loss of ASS1 leads to the overexpression of specific types of arginine transporters [[79]40]. Here, the transcript levels of several solute carriers (SLCs), such as Slc7a3, Slc7a5, Slc7a1 and Slc7a7 were assessed in mESCs. Notably, A26 and A27 mESCs showed remarkably higher levels of Slc7a1 and Slc7a3 transcripts (Fig. [80]1C). Notably, ASS1-knockout mESCs (A26 and A27) maintained dome-shaped colony morphology and alkaline phosphatase (AP) activity (Fig. [81]1D), and could be propagated beyond 45 passages while retaining clonogenicity. In addition, A26 and A27 mESCs demonstrated accelerated proliferation in growth curve assays (Fig. [82]1E). Although Ki67 staining revealed no significant change in the proportion of proliferating cells (Figure S1B), Annexin V/PI staining indicated reduced apoptosis in the A26 and A27 mESCs (Fig. [83]1F and G). These results indicate that the increased proliferation rate in the A26 and A27 mESCs is due to the reduction in apoptotic cells. Given the established link between cell cycle progression and proliferation, we performed cell cycle analysis. Flow cytometry revealed an increased G0/G1 phase fraction but substantially reduced S phase population in A26 and A27 versus W2 mESCs (Fig. [84]1H). As pluripotent stem cells commit to one of the three embryonic germ layers, their progeny acquire an extended G1 phase, resulting in increased cell division times [[85]41]. Therefore, the increase in the G1 phase in A26 and A27 mESCs might influence pluripotency and lineage differentiation. Collectively, ASS1 knockout preserves mESCs self-renewal and pluripotency while dysregulating arginine transporter expression and perturbing cell cycle progression. ASS1-knockout mESCs have pluripotent characteristic To further evaluate the pluripotency of mESCs, we performed immunocytochemistry, RT-qPCR, and Western blotting assays comparing control mESCs (W2) and ASS1-knockout mESCs (A26 and A27). Immunostaining confirmed Oct4 expression in both control (W2) and knockout mESCs (A26 and A27), while ASS1 was undetectable in A26 and A27 mESCs (Fig. [86]2A). RT-qPCR revealed a significant downregulation of pluripotency markers Oct4, Sox2 and c-Myc in A26 and A27 as compared with W2 mESCs (Fig. [87]2B). However, protein levels of pluripotency factors remained largely unchanged except for reduced Oct4 (Fig. [88]2C). Given this preserved pluripotent state, we examined lineage specification markers. A26 and A27 mESCs consistently upregulated mesendoderm markers (Mixl1 and Eomes) and mesoderm markers (T and Hand1) (Fig. [89]2D and [90]S1C), while downregulating ectoderm markers (Nestin and Otx2; Fig. [91]2E). In addition, the expression of endodermal markers in A26 and A27 mESCs was inconsistent when compared to W2 mESCs (Figure S1D). During embryonic development, a part of mesendoderm cells will differentiate into cardiac precursor cells and eventually into cardiomyocytes. Notably, the cardiomyocyte precursor marker Nkx2-5 was elevated in A26 and A27 mESCs (Fig. [92]2F). In vivo chimera assays further validated developmental competence. H2B-tdTomato-labeled A27 mESCs contributed extensively to E13.5 chimeric embryos across multiple tissues, including genital ridges. Furthermore, A27 mESCs were able to contribute to full-term chimeras (Fig. [93]2G, H and S1E). Five out of 12 full-term pups were chimeras, with a chimerism efficiency of 42% (Figure [94]S1F). Compared to non-chimeric mice, chimeric mice were smaller in size, and all chimeras died between 6 and 7 weeks. In summary, these results demonstrate that ASS1 knockout upregulates the expression of mesendoderm lineage genes and downregulates the expression of ectoderm genes in mESCs, without affecting their pluripotency in vivo. Fig. 2. [95]Fig. 2 [96]Open in a new tab ASS1 deficiency promotes mesendodermal gene expression in mESCs. (A) Immunofluorescence staining for pluripotency markers Oct4 and ASS1 in mESCs. Scale bars, 100 μm. (B) The relative mRNA levels of pluripotency markers in control (W2) and ASS1-knockout mESCs (A26 and A27) were measured via RT-qPCR. Values were normalized to β-Actin expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. p values were calculated by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Western blot analysis of pluripotency markers (Oct4, Nanog, Sox2 and c-Myc) and quantitative assessment of Oct4 protein levels in control (W2) versus ASS1-knockout mESCs (A26 and A27). β-Actin was used as the loading control. Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (D and E) Relative mRNA levels of mesendodermal and ectodermal markers. Values were normalized to β-Actin expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. p-values were calculated by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. (F) RT-qPCR analysis was conducted for Nkx2-5 in control (W2) and ASS1-knockout mESCs (A26 and A27). Statistically significant differences denoted as * p < 0.05 and ** p < 0.01 (Student’s t test). The data are presented as the mean ± SD of values from three independent experiments. (G) Germline contribution of ASS1-knockout A27 mESCs in gonads of chimeric (E13.5). Scale Bar, 100 µm. (H) Chimeric full-terms pups generated by injecting ASS1-knockout A27 mESCs into ICR host blastocysts ASS1-knockout mESCs have higher cardiomyocyte differentiation potential in vitro N2B27 medium (lacking LIF, 2i, and feeder cells) creates a permissive environment for autocrine factor-driven differentiation, enabling mESCs to exit pluripotency and undergo differentiation [[97]42, [98]43]. Subsequently, the expression levels of lineage-specific markers were examined. When cultured in cytokine-free N2B27, W2 mESCs exhibited extensive apoptosis during differentiation, whereas ASS1-knockout mESCs (A26 and A27) robustly formed beating cardiomyocyte clusters by day 10 (Fig. [99]3A and Video [100]1 and [101]2). Consistent with this phenotype, A26 and A27 mESCs showed significantly elevated mesendoderm marker expression during differentiation (Figure [102]S1G). To characterize the differentiation efficiency of beating cardiomyocytes, we counted the number of clones in which beating cardiomyocytes appeared on day 10 of differentiation. We found that the efficiency of beating cardiomyocyte differentiation in the A26 and A27 mESCs was significantly higher than that in the W2 mESCs (Fig. [103]3B). Consistent with this function, immunostaining confirmed robust expression of the cardiomyocyte marker cTnT in ASS1-knockout derivatives on day 12, whereas control cultures remained cTnT-negative (Fig. [104]3C). Since mesendoderm formation initiates cardiomyogenesis [[105]44], these findings demonstrate that ASS1 knockout enhances both mesendoderm commitment and subsequent cardiac lineage specification. Using cardiac differentiation, we observed comparable morphology between W2 and A27 mESCs (Fig. [106]3D and Video [107]3 and [108]4). To characterize the temporal evolution of cardiac maturation, cells from days 7, 9, 12, and 14 were collected and analyzed by RT-qPCR for expression of cardiac marker genes cTnT, a-Actin, Tbx5, Mef2c, Nkx2-5, and aMhc. Among them, the expression of α-Actin, Mef2c, Nkx2-5, and α-Mhc reached the peak on day 9. The expression of cTnT, Mef2c, Nkx2-5 and aMhc of A27 on day 9 was significantly higher than W2 mESCs. A27 mESCs exhibited significantly higher expression of α-Actin and Tbx5 than W2 mESCs on day 12 (Fig. [109]3E, F and [110]S1H-K). Notably, the mature cardiomyocyte marker Nkx2-5 and cardiomyocyte structural protein αMhc were still highly expressed in ASS1 knockout group (A27) compared to control group (W2) at day 23 of differentiation (Figure S2A). At differentiation day 12, cTnT immunostaining demonstrated positive cardiomyocyte formation in both control and ASS1-knockout groups (Fig. [111]3G). Critically, flow cytometry revealed a significantly higher proportion of cTnT^+ cells in ASS1-knockout group (A26 and A27) than in control group (W2) (Fig. [112]3H and I). One essential function of myocardial cells is the ability of beating. We investigated the difference in EBs beat properties between control EBs and ASS1 knockout EBs. The number, frequency and area of beating EBs during differentiation were counted. Notably, most of the EBs in the ASS1-knockout group (A27) demonstrated a beating phenomenon on day 7, and a large number of beatings EBs emerged on day 8. In contrast, only a small number of beating cardiomyocytes appeared from day 8 onwards in the control group (W2) (Fig. [113]3J). The frequency of beating cardiomyocytes in ASS1-knockout group (A27) was significantly higher than that in control group (W2) at days 12 and 14 (Fig. [114]3K). EBs in the ASS1-knockout group (A27) had a larger beating area than those in the control group (W2) (Fig. [115]3L). These results establish that ASS1 deficiency promotes mesendoderm specification and enhances the efficiency of cardiomyocyte differentiation and maturation. Fig. 3. [116]Fig. 3 [117]Open in a new tab The effect of ASS1 knockout in vitro differentiation. (A) The morphology of the control (W2) and ASS1-knockout cells (A26 and A27) was assessed on day 10 of N2B27 medium differentiation in vitro. Scale bars, 200 μm. (B) The number of beating clones was counted in control (W2) and ASS1-knockout group (A26 and A27) on day 10 of N2B27 medium differentiation in vitro. An initial seeding of 4000 cells per 48-well and 50 wells of differentiation per cell line was used in the experiment. * p < 0.05, ** p < 0.01 and *** p < 0.001 (Student’s t test). Data are presented as the mean ± SD of values from three independent experiments. (C) Immunofluorescence staining for cTnT in mESCs on day 12 of N2B27 medium differentiation. Scale bars, 100 μm. (D) Images of mESCs (W2) and ASS1-knockout group (A27) in days 2, 7, 9 and 12 of cardiac differentiation. Scale Bar, 200 µm. (E and F) RT-qPCR analysis of expression of cardiomyocyte marker genes Nkx2-5 and αMhc in the control (W2) and ASS1-knockout A27 mESCs on days 7-14 of cardiac differentiation. Data represented as mean ± SD from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). The expression levels were normalized by Gapdh. (G) Immunofluorescence images of mESCs stained for cTnT at Day 12 of cardiac differentiation. Scale bars, 100 μm. (H) Flow cytometry analysis of cTnT expression in the control (W2) and the ASS1-knockout mESCs (A26 and A27) on day 12 of cardiac differentiation. (I) The percentage of cTnT^+ cells in the control (W2) and the ASS1-knockout mESCs (A26 and A27) on day 12 of cardiac differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). Data are presented as the mean ± SD of values from three independent experiments. (J) Comparison of beating EBs numbers in control (W2) and ASS1 knockout group (A27) during cardiac differentiation in day 7-14. Data represented as mean ± SD from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (K) Frequencies of beating EBs for control (W2) and ASS1-knockout group (A27) in days 8-14 of cardiac differentiation. Data represented as mean ± SD from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (L) Beating area of EBs for control (W2) and ASS1-knockout group (A27) at day 9 of cardiac differentiation. Data represented as mean ± SD from three independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test) Transcriptome properties of ASS1-knockout mESCs To gain further molecular insights into ASS1-knockout mESCs, we performed RNA sequencing (RNA-seq) comparing control (W2) and ASS1-knockout (A27) mESCs. Differential expression analysis (fold change > 2, p < 0.05) identified 336 upregulated and 315 downregulated genes. Among downregulated transcripts, several genes involved in neuron differentiation and cellular nitrogen compound biosynthetic process were significantly reduced, including Stag3, Adssl1 and Nkx6-3. (Figs. [118]4A and [119]S2B). We next analyzed the expression of a series of markers that are typically associated with pluripotency, primitive streak/mesendoderm, early ectoderm and neuroectoderm. Primitive streak/mesendoderm markers, including for instance Eomes, T, and Mixl1, were highly enriched in A27 mESCs while pluripotency and early ectoderm and neuroectoderm markers were highly enriched in W2 mESCs (Fig. [120]4B). Subsequent assessment showed downregulation of both naïve-state and primed-state pluripotency genes in A27 mESCs (Figure S2C). Gene Ontology (GO) analysis of different expression gene sets indicated that these genes were associated with cellular nitrogen compound biosynthetic process, cell population proliferation, MAPK cascade, neuron differentiation and heart development (Fig. [121]4C). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed alterations in the alanine, aspartate, and glutamate metabolism, as well as enrichment of the MAPK cascade in differentially expressed gene sets (Figure S2D). On the other hand, to further understand the mechanism of metabolism and signaling pathways difference between A27 mESCs and W2 mESCs, Gene Set Enrichment Analysis (GSEA) was executed. The results indicated that, compared with W2 mESCs, the KO00250 alanine, aspartate and glutamate metabolic pathway decreased and KO04310 Wnt signaling pathway increased in A27 mESCs (Fig. [122]4D and [123]S2E). As expected, a series of Wnt cascade-related genes and aspartate metabolism-related genes observed by RT-qPCR were significantly increased in ASS1-knockout mESCs (A26 and A27) (Fig. [124]4E and S2F). Thereafter, we investigated a series genes of MAPK cascade in ASS1-knockout mESCs (A26 and A27) compared with those in control mESCs (W2) using RT-qPCR. No significant differences were observed in mRNA levels of MAPK cascade-related genes between control mESCs (W2) and ASS1-knockout mESCs (A26 and A27) (Figure S2G). Altogether, the evidence collected clearly suggests that ASS1 has key roles in controlling the cell signaling pathway, amino acid metabolism, and differentiation of mESCs. Fig. 4. [125]Fig. 4 [126]Open in a new tab Analysis of molecular features of ASS1-knockout mESCs. (A) Volcano plot illustrating the distribution of differentially expressed genes with a two fold change following ASS1 deletion. (B) Heatmap depicting the expression of representative markers in control (W2), ASS1-knockout mESCs (A27), including those associated with pluripotency, primitive streak/mesendoderm (PS/ME), and early ectoderm/neuroectoderm. (C) Gene Ontology (GO) analysis identifying significantly enriched "biological processes" (BP) of the differentially expressed genes (DEGs) in ASS1-knockout (A27) compared to control mESCs (W2). (D) GSEA (Gene Set Enrichment Analysis)-KEGG analysis of the transcriptome. (E) Validation of RNA-seq data was performed using RT-qPCR analysis to assess the relative mRNA levels of specified Wnt/β-catenin cascade-related genes in control (W2) and ASS1-knockout mESCs (A26 and A27). Values were normalized to Gapdh expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. p-values were calculated by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant ASS1 deficiency affects Wnt/β-catenin and JNK pathway in mESCs The impact of the function of Wnt/β-catenin signaling in the self-renewal of mESCs has been the subject of comprehensive research, and previous studies have suggested that Wnt/β-catenin signaling acts as a trigger of differentiation [[127]45–[128]48]. To confirm the activation of the Wnt/β-catenin pathway, we isolated the nucleus and cytoplasm of control (W2) and ASS1-knockout mESCs (A26 and A27), respectively, and assessed the relative expression of β-catenin in both compartments. We observed a significant increase in nuclear β-catenin levels, indicating activation of the Wnt/β-catenin pathway in ASS1-knockout mESCs (A26 and A27). However, we did not observe nuclear translocation of p-ERK (Fig. [129]5A). To investigate the potential impact of ASS1 loss on the Wnt/β-catenin pathway, we established two experimental groups using ASS1-knockout mESCs (A27): one cultured in 2i/LIF medium without CHIR99021 (GSK-3α/β inhibitor) (A27-LP), and another group treated with XAV939 (Tankyrase inhibitor) in A27-LP (A27-LPX). After 48 h, the expression of Eomes was assessed via RT-qPCR. Compared to the other group, Eomes expression was significantly reduced in A27-LPX, thereby indicating the involvement of Wnt/β-catenin signaling in differentiation into mesendoderm lineage in ASS1-knockout A27 mESCs (Fig. [130]5B). After that, the differential gene sets of Wnt pathway and MAPK pathway were correlated, and the transcription target genes were predicted by JASPAR database and MEME FIMO software. The results showed that Mafb and Jun family transcription factors may target multiple key node genes in Wnt and Mapk pathway (Fig. [131]5C). Given that extracellular signal-regulated kinase (ERK) signaling is a driving force for mESCs differentiation and that ERK also crosstalk with JNK signaling. Moreover, studies show that JNK signaling is required for lineage-specific differentiation but not stem cell self-renewal. In human embryonic stem cells (hESCs), c-Jun as a transcription factor downstream of JNK signaling, can block cardiomyocyte differentiation by regulating histone modifications and altering chromatin accessibility [[132]49–[133]52]. Western blot analysis revealed significantly reduced p-JNK and c-Jun protein levels in ASS1-knockout mESCs (A26 and A27) (Fig. [134]5D). Since arginine functions as an epigenetic modulator through interactions with TEAD4, STAT3, and histone modification [[135]27], we assessed whether ASS1-mediated arginine biosynthesis impairment alters chromatin landscapes. Western blot analysis demonstrated that H3K27me3 and H3K9me3 were increased in A26 and A27 mESCs (Fig. [136]5E). Transcriptome analysis further identified differential expression of histone-modifying enzymes in control (W2) and ASS1-knockout (A27) mESCs (Fig. [137]5F). In order to exclude the effect of off-target, we opted to overexpress ASS1 in ASS1-knockout mESCs (A27). Specifically, we introduced pCMV-ASS1-Neomycin into A27 mESCs via electroporation and allowed the cells to be cultured for 48 h (Fig. [138]5G). Subsequently, we assessed the expression levels of pluripotency, mesendoderm, and ectoderm markers in these cells. Our results demonstrated that the overexpression of ASS1 in A27 mESCs led to a significant decrease in the expression of mesendodermal markers (Eomes, Mixl1), as well as an increase in pluripotency markers (Sox2) and ectoderm markers (Nestin, Otx2) (Fig. [139]5H). This suggests that the observed phenotype was effectively rescued. In summary, our research indicates that depletion of ASS1 in mESCs leads to the upregulation of mesendoderm related genes. Additionally, we have observed loss of ASS1 affects the expression of c-Jun of mESCs and activates the Wnt/β-catenin pathway. Treatment of ASS1-knockout mESCs with Wnt/β-catenin pathway inhibitors can reverse the mesendoderm bias. However, further investigation into the specific targets of ASS1 that are linked to the Wnt/β-catenin pathway is necessary to gain a deeper understanding of its mechanism. Fig. 5. [140]Fig. 5 [141]Open in a new tab ASS1 deficiency affects the activation of signaling pathways in mESCs. (A) The effect of ASS1 knockout on ERK signaling pathway and Wnt/β-catenin in mESCs were detected by Western blotting. GAPDH and H3 served as a loading control. Quantitative analysis of β-catenin protein levels in control (W2) and ASS1-knockout mESCs (A26 and A27). Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (B) Expression of Eomes at the RNA level was assessed in W2 and A27 mESCs under 2i/LIF culture, as well as in A27 mESCs treated with LIF + PD (LP) or LIF + PD + XAV939 (LPX) for 48 h. The experiments were performed in triplicate, and error bars represent the SD. The expression levels were normalized by β-Actin. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (C) Transcription factor target genes regulatory network diagram. The shape and size of the nodes represent the connectivity. Transcription factors are represented by circles and target genes by triangles. (D) Quantitative analysis of p-JNK and c-Jun protein levels in control (W2) and ASS1-knockout mESCs (A26 and A27). β-Actin served as a loading control. Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (E) Quantitative analysis of H3K9me3 and H3K27me3 protein levels in control and ASS1-knockout mESCs. H3 served as a loading control. Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test). (F) Heatmap of histone modification related genes expression patterns in control (W2) and ASS1-knockout A27 mESCs. (G and H) Relative mRNA levels of pluripotency, ectoderm and mesendoderm markers in A27 mESCs electroporated with Neomycin control vector and ASS1-Neomycin overexpression vector. The expression levels were normalized by β-Actin. Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t test) Discussion Our findings reveal a critical role for ASS1 in mouse embryonic stem cell fate decisions. The progressive reduction of ASS1 expression from mESCs, EpiSCs, XEN, and MEFs lineages suggests a coordinated metabolic reprogramming that accompanies cellular specialization. This observation might be attributed to the increased reliance of the extracellular arginine, while intracellular arginine synthesis activity diminishes during the cell differentiates. Notably, ASS1 knockout did not compromise mESCs pluripotency. The ASS1-knockout mESCs maintained normal pluripotency marker expression and retained germline transmission capacity, as evidenced by successful chimera generation. ASS1 acts as a metabolic checkpoint that, following DNA damage, blocks cell cycle progression by restricting nucleotide synthesis and p53-related gene transcription, thereby achieving genomic maintenance and cell survival [[142]53]. The experimental findings also revealed that ASS1 knockout in mESCs leads to altered proliferation and cell cycle progression. In this study, only the synthesis pathway of arginine was blocked in the cells, and arginine in the culture environment might maintain the pluripotency of mESCs. This result is also consistent with the previous report showed that the concentration of arginine in the culture medium can notably impact the proliferation of trophectoderm [[143]54, [144]55]. The mESCs progressively exit pluripotency and differentiate in N2B27 medium in the absence of feeder cells, 2i, and LIF [[145]43, [146]56, [147]57]. Therefore, it will be interesting to compare the differentiation characteristics of ASS1 knockout and control mESCs in the N2B27 medium. Notably, we observed a significant increase in the proportion of beating cardiomyocytes in ASS1-knockout mESCs compared to control mESCs in N2B27 medium. Recently, it has been demonstrated that loss of ASS1 impairs neuronal differentiation in zebrafish [[148]58]. In our study, we also observed that ASS1 knockout led to decreased expression of ectoderm markers in mESCs. Meanwhile, the observed upregulation of mesendoderm in N2B27 medium indicates that ASS1 knockout not only promotes mESCs differentiation toward the mesodermal lineage in vitro, but also reveals a synergistic interaction between specific components of this defined medium and ASS1 in driving the progression of cardiomyocytes differentiation. In addition, we also used conventional method to induce cardiomyocytes in control mESCs and ASS1-knockout mESCs, the results strongly support differentiation of ASS1-knockout mESCs in N2B27 medium. To our knowledge, cardiac markers such as Nkx2-5 and Mef2c are act as hypertrophic activating factors in the heart [[149]59]. During the differentiation of cardiomyocytes, our data indicated significant enhancement in the expression of these markers upon the knockout of ASS1. It is noteworthy that during cardiomyocyte differentiation, the ASS1 knockout group exhibited a beating frequency closer to physiological conditions and a larger beating area. Additionally, from day 9 to day 23 of differentiation, the expression of Nkx2-5 —a marker gene for mature cardiomyocytes [[150]60]—was significantly higher in ASS1-knockout group compared to the control group. This may suggest that the absence of ASS1 could facilitate more efficient differentiation of mESCs into adult cardiomyocytes with normal physiological functions. In our study, we demonstrated that ASS1-knockout mESCs exhibit increased propensity for cardiomyocyte formation. This indicates that ASS1 might serve as a negative regulator during cardiomyocyte development. There are several evidences indicating that exogenous signals initiated by growth factors play a pivotal role in the differentiation of mESCs into mesendoderm [[151]61–[152]64]. Previous studies have shown the participation of the ERK1/2 signaling pathway and Wnt/β-catenin pathway in the differentiation of mESCs into mesendoderm [[153]65–[154]68]. Here, we observed observed an enrichment of Wnt pathway-related genes, up-regulation of key proteins, and nuclear translocation of β-catenin in ASS1-knockout mESCs. The essential role of the Wnt/β-catenin pathway in regulating embryonic stem cell pluripotency and specification has been extensively documented [[155]69–[156]71]. Hence, we investigated whether Wnt/β-catenin pathway is involved in ASS1-knockout mESCs. The result demonstrated ASS1-knockout mESCs responsiveness to Wnt inhibitor supplemented medium, and exhibited downregulated Eomes in culture upon treatment. Our findings parallel recent work in human pluripotent stem cells: Yuan et al. established an ASS1 heterozygous knockout hESC line (WAe001-A-13) that retains pluripotency despite impaired urea cycle function [[157]72]. The conserved maintenance of stemness in both human and murine ASS1-deficient systems suggest a fundamental role of ASS1 in regulating metabolic-epigenetic balance rather than core pluripotency circuits. However, our discovery that ASS1 ablation actively promotes mesendoderm/cardiomyocyte differentiation in mESCs through Wnt/β-catenin hyperactivation reveals a previously unrecognized developmental function. Recent studies have indicated that down-regulation of c-Jun, which was a downstream target protein of the JNK pathway, causes embryonic stem cells to differentiate along mesoderm and endoderm lineages, and eventually develop into cardiomyocytes [[158]52, [159]73]. Consistent with these findings, our data also showed that c-Jun and p-JNK is significantly down-regulated in ASS1-knockout mESCs. Therefore, it is possible that the downregulation of ASS1 may affect the JNK pathway through an unknown mechanism, ultimately leading to an increase in the efficiency of mESCs differentiation into cardiomyocytes. Conclusions Our study effectively recapitulated the significance of ASS1 in early mammalian development using mESCs. We demonstrated that ASS1 deficiency redirects differentiation potential toward mesendoderm while suppressing ectodermal commitment, culminating in enhanced cardiomyocyte generation efficiency. During this process, the Wnt signaling pathway and c-Jun serve as key regulatory hubs. ASS1 is a rate-limiting enzyme present in the cytoplasmic arginine synthesis pathway known to exert its function by regulating cellular arginine levels in various biological contexts. We propose that ASS1 plays a crucial role in regulating arginine synthesis and related metabolic pathways, while also fine-tuning mESCs fate decisions toward the cardiomyocyte lineage. These findings broaden the scope for future investigations and suggest ASS1 as a promising candidate target for investigating novel therapies against heart disease. Supplementary Information [160]13287_2025_4622_MOESM1_ESM.pdf^ (2.4MB, pdf) Additional file 1 Fig. S1 ASS1 deficiency promotes mesendoderm lineage-specific gene expression. (A) ASS1-knockout mESCs were generated via CRISPR-Cas9 genome editing as illustrated. (B) Representative flow cytometry plots of Ki-67 staining of control (W2) and ASS1-knockout mESCs (A26 and A27) on day 5 post seeding to study the cell proliferation status. (C and D) RT-qPCR analysis of endoderm and mesoderm-associated genes expression in the ASS1-knockout mESCs (A26 and A27), with W2 mESCs used as the control. Data were obtained in triplicate and presented as mean ± SD. p-values were calculated by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. The expression levels were normalized by β-Actin. (E) ASS1-knockout A27 mESCs contribute to various tissues of the E13.5 chimeric embryo. Scale Bar, 200 µm. (F) The proportion of chimeric mice (n=5) among newborn mice (n=12). (G) The relative mRNA expression of mesendodermal markers in control (W2) and ASS1-knockout mESCs (A26 and A27) were analyzed via RT-qPCR at day 10 after N2B27 medium differentiation. The expression levels were normalized by β-Actin. Statistical analysis was conducted using two-way ANOVA, with * p < 0.05, ** p < 0.01, and *** p < 0.001 indicating significance. Data are presented as the mean ± SD of values from three independent experiments. (H-K) RT-qPCR analysis of cardiomyocyte marker genes expression (Mef2c, cTnT, α-Actin, Tbx5) in the W2 mESCs and ASS1-knockout A27 mESCs in days 7-14 of cardiac differentiation. Data represented as mean ± SD from three independent experiments, * p< 0.05, ** p < 0.01, *** p< 0.001 (Student’s t test). The expression levels were normalized by Gapdh. Fig. S2 Molecular properties of ASS1-knockout mESCs. (A) RT-qPCR analysis expression of cardiomyocyte marker genes (Nkx2-5, Mef2c, αMhc, α-Actin, Tbx5) in the W2 mESCs and ASS1-knockout A27 mESCs on day 23 of cardiac differentiation. The expression levels were normalized by Gapdh. Data were obtained in triplicate and presented as mean ± SD. * p < 0.05, ** p< 0.01, *** p< 0.001 (Student’s t test). (B) The differential gene statistics map counted the genes with 2-fold difference after ASS1 knockout. Abscissa: pairwise comparison of samples; Ordinate: number of differentially expressed genes; Yellow represents up-regulated differentially expressed genes. Blue represents the down-regulated differentially expressed genes. (C) Heatmap of naïve and primed-related genes expression patterns in control (W2) and ASS1-knockout A27 mESCs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was performed on the differentially expressed genes (DEGs) for ASS1-knockout A27 mESCs versus W2 mESCs. (E) GSEA (Gene Set Enrichment Analysis)-KEGG analysis of the transcriptome. (F) RT-qPCR analysis of transcript levels of alanine, aspartate and glutamate metabolic pathway-associated genes in control (W2) and ASS1-knockout mESCs (A26 and A27). Values were normalized to β-Actin expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. p values were calculated by two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001. (G) Validation of RNA-seq data was performed using RT-qPCR analysis to assess the relative mRNA levels of MAPK cascade-related genes in control (W2) and ASS1-knockout mESCs (A26 and A27). Values were normalized to β-Actin expression and relative expression was plotted by assaying three biological replicates. Error bars indicate mean ± SD. p values were calculated by two-way ANOVA, * p < 0.05, ** p< 0.01, *** p < 0.001. [161]Additional file 2.^ (13KB, xlsx) [162]Additional file 3.^ (13.9KB, xlsx) [163]Additional file 4.^ (1.5MB, avi) [164]Additional file 5.^ (636.4KB, avi) [165]Additional file 6.^ (1.7MB, avi) [166]Additional file 7.^ (2.9MB, avi) Acknowledgements