To investigate the potential impact of Tet deletion on cellular metabolism, we performed a series of metabolic flux assays using both wildtype (WT) and Tet-triple knockout (Tet-TKO) mESCs. The Tet-TKO mESCs were generated as previously reported utilizing a CRISPR-Cas9-based genome editing approach (Fang et al., 2019). We selected three independent clones of Tet-TKO mESCs and confirmed deletion efficiency through locus-specific Sanger sequencing and western blot analysis (Fang et al., 2019). All three clones showed a complete absence of detectable 5-hydroxymethylcytosine (5 hmC) as a major catalytic product of Tet enzymes. To investigate the impact of Tet deletion on cellular energetics, we assayed oxidative phosphorylation and glycolysis activity in WT and Tet-TKO mESCs by measuring oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), respectively. Mitochondrial stress tests revealed similar oxidative phosphorylation activity between WT and Tet-TKO mESCs (Figure 1A). Tet-TKO mESCs displayed significantly higher ECAR rates than the WT mESCs in a glycolysis stress test, indicating increased glycolysis activity (Figure 1B). Cells were cultured under non-glycolytic conditions to establish a baseline. Subsequent glucose treatment allows calculation of the rate of glycolysis. Oligomycin was added to inhibit oxidative phosphorylation and drive the full capacity of glycolysis, allowing determination of the glycolytic capacity. Finally, cells were exposed to 2-Deoxy-D-glucose (2-DG), a glucose analog that is inefficiently metabolized through glycolysis. The 2-DG treatment evaluates the cellular response to energy demands, enabling the determination of the glycolytic reserve value, or cellular capability to shift energy production towards glycolysis. The Tet-TKO clones exhibited a 1.2 to 2.1-fold increase in the glycolysis rate, glycolytic capacity, and glycolytic reserve ability when cells were successively challenged with glucose, oligomycin, and 2-DG (Figure 1C). Additionally, we quantified the glycolytic rates in WT and Tet-TKO mESCs by exposing cells to rotenone/antimycin A (Rot/AA), followed by 2-DG treatment (Figure 1D). In this assay, Rot/AA is used to inhibit mitochondrial function and measure mitochondria-associated acidification. The use of Rot/AA and 2-DG treatment allows the differentiation between acidification from mitochondrial oxidative phosphorylation and aerobic glycolysis. Our results revealed elevated levels of basal and compensatory glycolysis upon Tet deletion (Figure 1E), suggesting a higher proportion of proton efflux derived from glycolysis in Tet-TKO mESCs compared to WT mESCs (Figure 1F).

ATP, which is derived from both mitochondrial OXPHOS and cytosolic glycolysis, serves as a major energy source to support cell survival and growth. To assess the impact of Tet deletion on ATP production, we calculated ATP production based on the OCR and ECAR data obtained from WT and Tet-TKO mESCs. While the overall basal ATP production rate appeared similar across all groups (Figure 1G), Tet deletion resulted in a higher proportion of ATP being generated from glycolysis compared to OXPHOS (Figures 1H, I). Collectively, these findings indicate that Tet deficiency leads to aberrant glucose metabolism in mESCs.

High glycolytic flux is a common characteristic observed in various stem cells, including embryonic stem cells. To assess the impact of Tet deletion on glucose uptake, we treated WT and Tet-TKO mESC clones (one and two) with 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose), a fluorescent glucose analog that allows for direct measurement of glucose uptake using flow cytometry. We observed an approximately 1.7-fold increase in 2-NBDG signals in Tet-TKO mESCs compared to the WT group (Figure 2A), suggesting enhanced glucose uptake following Tet deletion. In parallel, we employed mass spectrometry analysis to measure the intracellular pyruvate and lactate levels in the same cells. Consistent with 2-NBDG uptake results, we found elevated levels of both pyruvate and lactate in Tet-TKO mESCs compared to WT cells (Figure 2B). The increase of lactate uptake is also observed in cells lacking SIRT6, an NAD + -dependent histone deacetylase (Zhong et al., 2010). The increased intracellular lactate level in Tet-TKO cells was further independently validated by lactate colorimetric assay (Figure 2C). Considering that elevated intracellular lactate has been reported to alter the intracellular pH level (Miyazawa et al., 2022), we introduced a ratiometric pH sensor, GW1-pHRed, into WT and Tet-TKO mESCs. Tet-TKO mESC showed a more prominent increase in the fluorescence intensity ratio of the pH sensor, indicating a lower pH in the cytosol (Figure 2D). Overall, these results indicate enhanced glucose uptake, higher levels of pyruvic acid and lactate, and disrupted intracellular pH regulation in Tet-TKO mESCs, supporting the role of Tet genes in modulating stem cell metabolism.

To gain further insights into the mechanism underlying increased glucose uptake in Tet-TKO mESCs, we examined the transcription of key genes involved in glucose uptake using real-time quantitative PCR (qPCR). Our analysis revealed a significant upregulation of Slc2a1 (Glut1), Ldha, and Slc16a1 (Mct1) genes in Tet-TKO clones compared to WT cells (Figure 2E). Glucose transporter 1 (Glut1) is a transporter protein directly involved in the uptake of glucose into cells (Olson and Pessin, 1996). Lactate dehydrogenase A (Ldha) is an enzyme involved in the metabolism of L-lactate and pyruvate (Chung et al., 1985). Monocarboxylate transporter 1 (Mct1) encodes a proton-coupled symporter called that facilitates lactate transportation (Garcia et al., 1994). Collectively, these findings provide valuable insights into the molecular changes associated with Tet deletion and the potential mechanisms underlying the observed increase in glucose uptake and metabolism in Tet-deficient mESCs.

To investigate whether elevated glucose intake and metabolism in Tet-TKO mESCs alter cellular function, we measured cell proliferation and viability under different glucose concentrations (high: 4.5 g/L, low: 1.0 g/L, no: 0 g/L). No significant difference in cell growth was observed between WT and Tet-TKO mESCs under these conditions (Figure 3A). Additionally, qPCR analysis revealed no significant changes in the expression levels of Nanog and Oct4, key genes involved in stem cell pluripotency (Figure 3B). These data suggest that increased glucose metabolism resulting from Tet deficiency has minor impacts on the proliferation and pluripotency of mESCs.

Given that increased glucose levels can lead to upregulation of acetyl-CoA and alter histone acetylation, we measured the intracellular acetyl-CoA level and the transcription of key genes involved in acetyl-CoA metabolism (Figures 3C, D). Our data suggests that increased intracellular glucose levels in Tet-TKO mESCs do not exert significant effects on acetyl-CoA levels or the expression of Acly and Accs2, two key genes involved in acetyl-CoA production (Wellen et al., 2009; Mews et al., 2017). Furthermore, the histone acetylation level remains unaffected in Tet-deficient mESCs (Figures 3E, F).

Next, we conducted additional investigations to determine the impact of different glucose concentrations on the differentiation capability of Tet-TKO mESCs. To assess this, we performed embryoid body (EB) formation assays using WT and Tet-TKO mESCs at varying glucose levels: high (4.5 g/L), low (1.0 g/L), and no glucose (0 g/L). Similar to the undifferentiated cells, the differentiated Tet-TKO embryoid bodies exhibited significantly higher ECAR than that of WT embryoid bodies in a glycolytic rate test (Figure 4A). Consistent with previous research (Koh et al., 2011; Fang et al., 2022), Tet-TKO mESCs showed a significant reduction in the ability to form embryoid bodies under all conditions in a dose-dependent manner, impacting both the size and number of EBs without affecting cell viability (Figures 4B–D). Interestingly, WT mESCs showed only minor changes in response to reduced glucose conditions (Figures 4B, C). In fact, the relative EB size of Tet-TKO mESCs cultured under high glucose conditions was significantly lower than the EB size of WT-TKO mESCs under low and no glucose conditions (Figure 4E). To gain further insights, we measured the expression of genes crucial for glucose metabolism in differentiated WT and Tet-TKO cells under different glucose concentrations. Similar to undifferentiated mESCs, Tet-TKO embryoid bodies continued to exhibit higher expression of genes involved in glucose metabolism, such as Glut1, Ldha, and Mct1, under high-glucose culture conditions (Figure 4F). Interestingly, a significant reduction in the expression of these genes was detected in Tet-TKO, but not WT, embryoid bodies under reduced glucose culture conditions compared to the high glucose culture condition (Figure 4F). In summary, these findings indicate that Tet-deficient mESCs rely more heavily on glucose levels compared to WT cells during differentiation.

Mitomycin C treated mouse embryonic fibroblasts (MEF) were used as feeder cells for mESCs culture in this study. Normal culture medium is the Knockout Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 15% fetal bovine serum (Omega), 1.0% penicillin-streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM 2-mercaptoetanol (Sigma), and 103 U/mL of leukemia inhibitory factor (LIF; Millipore). Various glucose treatment mediums consisted of DMEM without glucose supplemented with 15% fetal bovine serum (Omega), 1% penicillin-streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Sigma), 103 U/mL of leukemia inhibitory factor (LIF; Millipore), and D-(+)-glucose solution 45% at varying concentrations (high: 4.5 g/L; low: 1.0 g/L; and no: 0 g/L; Corning).

EB formation is performed using the hanging drop culture method. mESCs are suspended into single cells and then resuspended in EB formation media (mESC culturing medium without LIF). The cells are then suspended in a dish pre-coated with 0.2% gelatin and incubated at 37°C with 5% CO2 for 30 min to remove MEF. mESCs are resuspended at 500 cells per 20 µL (20 µL/drop) containing varying glucose concentrations and inoculated onto the lid of the tissue culturing dish. The lid is then inverted and placed on top of the dish containing 15 mL of PBS and cultured for 5 days. Afterward, the EBs are collected for further analysis. For the cell viability assay, the EBs are washed once with PBS and then trypsinized into single cells. The 7-AAD Viability Staining Solution (Biolegend) followed by flow cytometry analysis is performed to check the cell viability.

Mouse ESCs were seeded in XF96 cell culture microplates (Agilent) coated with 1.5% fibronectin at a density of approximately 20,000 cells per well in normal culture medium, and then incubated overnight. The following day, the Seahorse Glycolytic Rate Assay, Seahorse Glycolysis Stress Test, Seahorse Mito Stress Test, or ATP Real-Time Rate Assay were performed on the XF96 Analyzer (Agilent) according to the manufacturer’s instructions. Seahorse results were normalized to cell counts. The normalized Seahorse data were analyzed using Wave, the software for the XF Analyzer from Agilent.

Mouse ESCs were harvested, and DNA-free RNA was isolated using the NucleoSpin RNA Plus kit (MACHEREY-NAGEL), as per the manufacturer’s manual. Purified total RNA (500 ng) was reverse transcribed into cDNA using the PrimeScript RT Master Mix (TaKaRa). Real-time quantitative PCR was performed on a QuantStudio3 (Thermo Fisher Scientific) instrument using the 2X Universal SYBR Green Fast qPCR Mix (ABclonal). A three-step cycling program was employed, which included an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s. The mRNA expression of the target gene was normalized to 18S. All the primers were synthesized by Integrated DNA Technologies.

mESCs were rinsed with PBS twice and lysed in a radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% TritonX-100, 0.1% SDS, 0.5% Sodium deoxycholate, 50 mM Tris-HCl pH8.0), supplemented with Xpert Protease Inhibitor Cocktail Solution (100X) (GenDEPOT) on ice for 15 min. Cell debris was removed by centrifugation at 12,000 x g at 4°C for 15 min. For histone extraction, mESCs were rinsed with PBS twice and lysed in Triton extraction buffer (TEB: PBS containing 0.5% Triton X-100 (v/v), 2 mM phenylmethyl-sulfonylfluoride (PMSF), 0.02% (w/v) NaN3) on ice for 15 min. The lysate was then centrifuged at 12,000 x g at 4°C for 15 min, and the supernatant was removed and discarded. The pellets were then washed with TEB twice and resuspended in 0.2 N HCl overnight at 4°C. The lysates were then centrifuged at 12,000 x g at 4°C for 15 min, and the supernatant was collected for western blot analysis. The cell lysate was loaded onto 10% or 14% SDS-PAGE gels after mixing with SDS loading buffer (100 mM Tris-HCl, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM DTT, pH 6.8) and denaturing at 100°C for 10 min. Proteins were transferred onto Nitrocellulose membranes (Millipore) and blocked in 5% Bovine Serum Albumin (GenDEPOT) for 1 h at room temperature. Membranes were then incubated with the primary antibodies listed below at 4°C overnight, followed by incubation with a secondary antibody at room temperature for 1 h. West-Q Pico Dura ECL Solution (GenDEPOT) was used for development, and the antigen–antibody complexes were detected using the ChemiDoc Imaging system (Bio-Rad). Antibodies used were: Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling #7074), anti-rabbit IgG, HRP-linked Antibody (Cell Signaling #7076), anti-Histone H3 (Abcam, ab1791), anti-Histone H3 (acetyl K27) (Abcam, ab4729), anti-β-Actin (ABclonal, AC038), anti-GAPDH (ABclonal, AC027), and anti-acetyl lysine (Sigma-Aldrich, AB3879).

mESCs were seeded in a 12-well cell culture plate with a final volume of 1.5 mL of normal culture medium per well, and then incubated under standard cell culture conditions overnight. Subsequently, the cells were rinsed with PBS and incubated with normal culture medium for 2 h. Cell pellets were then collected and tested for lactate levels following the manufacturer’s instructions for the Lactate Assay Kit (MAK064, Sigma-Aldrich) or for acetyl-CoA levels following the instructions for the Acetyl-Coenzyme A Assay Kit (MAK039, Sigma-Aldrich).

The GW1-pHRed (Tantama et al., 2011) plasmid was purchased from Addgene (#31473). mESCs were transfected with pHRed using the iMfectin DNA transfection reagent (Gendepot). The pHRed signal was captured using the W1-Yokogawa/Nikon Live cell Imaging Spinning Disk confocal microscope. pHRed was excited using dual peaks at 560 and 430 nm, and its emission peak was at 610 nm. ImageJ was utilized for the measurement and quantification of histological data.

A FACS-based glucose uptake assay was conducted according to a previously published method (Dong and Alahari, 2018). Briefly, mESCs were seeded in a 24-well cell culture plate with a final volume of 1.0 mL of normal culture medium per well, and then incubated under standard cell culture conditions overnight. Thereafter, the cells were rinsed with PBS and incubated with 1.0 mL of culture media with varying glucose concentrations for 24 h. Next, the cells were rinsed with PBS and incubated with 1.0 mL of culture media containing various glucose concentrations, supplemented with 100 µM 2-NBDG for 2 h. The cell pellets were then collected and kept in ice-cold FACS buffer (PBS with 2% FBS) for analysis on a BD Biosciences LSRII analytical flow cytometer. The mean fluorescence intensities were analyzed using the FlowJo software.

A FACS-based cell viability assay was conducted according to the manufacturer’s instructions. Briefly, LIVE/DEAD Fixable Violet stain (Invitrogen) was diluted by adding 50 µL of DMSO to the vial. Mouse ESCs were seeded in a 24-well cell culture plate with a final volume of 1.0 mL of normal culture medium per well, and then incubated under standard cell culture conditions overnight. Thereafter, the cells were rinsed with PBS and incubated with 1.0 mL of culture media with varying glucose concentrations for 24 h. The cell pellets were then resuspended in 1 mL of FACS buffer with 1 µL of diluted live/dead dye, followed by a 30-min incubation at room temperature. The cells were washed twice with FACS buffer before analysis on a BD Biosciences LSRII analytical flow cytometer. The gating and analysis of the percentages of viable or dead cells were performed using the FlowJo software.